Cell Cultures: Types, Isolation, and Growth PDF

1 CELL CULTURES Cell cultures are a set of laboratory techniques used to isolate and maintain cells in arti cial laboratory conditions. It is possible to isolate and culture cells from any organism, though some are more dif cult than other...

1 CELL CULTURES Cell cultures are a set of laboratory techniques used to isolate and maintain cells in arti cial laboratory conditions. It is possible to isolate and culture cells from any organism, though some are more dif cult than others. Once isolated, the cells can be kept in arti cial conditions by controlling temperature, pH, etc. This allows the cells to proliferate and maintain the culture for a certain period of time. TYPES OF CELL CULTURES Cell cultures are divided into two main types: Primary Cultures: These are obtained directly from a tissue. They correspond to cells isolated from a speci c organism of interest. Cell Lines: Cell lines are cultures of cells that already exist in the lab and have the ability to divide, usually for a nite period, but sometimes inde nitely. There are nite, continuous cell lines, and cell strains. ◦ Finite Lines: These are cell lines that, after a certain number of divisions, enter senescence, causing the culture to die. ◦ Continuous Lines: In continuous lines, the capacity for division is in nite – the cells are immortal. These lines are derived from tumor biopsies or through transformation, which can be spontaneous due to mutations or induced by chemicals, viruses, etc. Any "normal" cell has a nite number of controlled and determined divisions. However, certain errors during the cell cycle result in mutations in genes that either prevent cell death or control cell proliferation, causing the cells to keep proliferating. These types of mutations are not detected by the cell’s error control mechanisms and occur, for example, in tumors. ◦ Cell Strain: The term "cell strain" is typically used when a speci c subpopulation is selected from a cell line. For example, one may want, in a culture of hepatocytes, cells that only express a particular marker. Through a set of techniques, it is possible to isolate these cells and have a genetically very similar culture, as there are differences among individual components of cell cultures, and growth conditions may increase or decrease this variability. CELL ISOLATION It is possible to isolate cells from any animal or plant. However, once the target tissue is selected, a cell extraction protocol must be implemented and optimized, as different cells have varying needs and forms of interaction. Cell isolation is usually done through a mechanical process combined with enzymatic digestion. The separated and isolated cells are placed in a culture medium, thus creating the primary culture. The cells in the culture begin to grow and proliferate until they cover the entire surface of the Petri dish. At this point, a portion of the primary culture is transferred to a new culture medium, giving rise to a cell line. CELL GROWTH When the cells are isolated, they are seeded in a Petri dish (primary culture) with a culture medium, and conditions similar to in vivo, such as temperature and pH, are simulated. In the rst few days after seeding, the initial number of cells decreases as some die due to damage during isolation. After these rst days of decline, the cells begin to proliferate until they ll the culture dish. At this point, the rst subculture is performed into a new dish, and the cells begin to grow exponentially in the original dish, requiring several subcultures (which generate cell lines) at speci c intervals. When they reach the limit of divisions, nite lines rapidly decrease in number, whereas continuous lines perpetuate their growth. fi fi fi fi fi fi fi fi fi fi fi fi fi fi fi fi  2 CELL CULTURES Cell cultures can grow in solid/semi-solid or liquid media. In solid media, the cells grow in a monolayer organization, while in liquid media, they grow in suspension. In monolayer growth, the limiting factor is space, whereas in suspension growth, the limiting factor is density. CELL MORPHOLOGY There are three types of cell morphologies, and depending on the morphology, they can grow in different types of cultures: Fibroblastic: These cells are bipolar or multipolar with elongated shapes and grow in monolayers, attached to a substrate. Epithelial: Epithelial cells have polygonal shapes and more regular dimensions, also growing attached to a substrate. Lymphoblastic: These cells have a spherical shape and generally grow in suspension. PRIMARY CULTURES vs. CELL LINES Primary Cultures Advantages: Cells retain many in vivo characteristics and high differentiation. Cells maintain high biotransformation capacity and enzymatic activity. Disadvantages: The initial set of cells obtained is very heterogeneous. Preparing these cultures is labor-intensive. Each experiment requires a new isolation.  3 The lifespan of these cultures is limited. Cells may contain pathogens. Cell Lines Advantages: Cultures can be frozen in liquid nitrogen and used from stock when needed, providing in nite proliferation (different from immortal cells). They have greater genotypic and phenotypic homogeneity. They offer high availability and growth capacity. They are free of pathogens. They provide consistent results. Disadvantages: They lose many characteristics present in the in vivo tissue, such as losing biotransformation enzymes. USES OF CELL CULTURES Cell cultures can be used for regulatory purposes, such as in ecotoxicology, allowing the determination of toxicological pro les, lethal doses, etc., and reducing the number of animals used. There are two main areas for using cell cultures: Research: Used as models instead of animals, for toxicity testing (including cytotoxicity), drug development, cancer research, genetic engineering, gene therapy, etc. Industry: In industry, cell cultures are used for the synthesis of compounds or tissues. They are employed to produce proteins, vaccines, to replace tissues or organs, for cellular agriculture, etc. SOURCES OF CELL LINES Currently, there are several cell line banks, such as ATCC, ECACC, DSMZ, where various cell lines from different organisms can be acquired, including sh, whose lines are still not very common. Alternatively, cell lines can be obtained through research and collaboration with other scientists. However, these collaborations can bring additional problems because donated cell cultures may not be what they claim to be. Morphologically similar cells can often be wrongly donated. Around 4,000 human cell lines are available, but only 400 to 500 sh lines, many of which are not commercially available. Nevertheless, in 2019, there were about 3.3 times more publications involving sh cell lines on PubMed than in 2000. LABORATORY CONDITIONS FOR WORKING WITH CELL CULTURES Two aspects must be considered: biosafety and the materials and equipment needed for the work. WHO De nition of Biosafety: A set of practices necessary to avoid accidental exposures to the environment, the operator, and the object. There are different levels of biosafety: BSL-1: The lowest risk, posing no risk to the operator or the environment. BSL-2: There is some risk, though minimal, to the operator and the environment when handling these materials, which include most cell cultures. It requires an isolated room, lockable doors, authorized entry only for experienced personnel, minimal entry and exit, laminar ow hoods placed away from doors or windows, etc. All waste must be autoclaved. BSL-3 and BSL-4: These are higher levels of biosafety depending on the type of cultures used. Equipment fi fl fi fi fi fi fi  4 Laminar Flow Hoods: There are three types – Class I, II, and III. Class I protects the operator and the environment but not the material handled. Class II protects the user, environment, and material. Class III provides almost total isolation of the internal and external environment. Inside these hoods, the air is sterilized using HEPA lters and air ow circulation. Incubators: These are used to create an optimal environment for cell growth. For example, human cell lines require higher temperatures (37°C), while sh lines need lower temperatures. CO₂ concentration is also crucial for some cultures. Inverted Microscopes: These have the light source on top and the objectives below the sample, as cells grow at the bottom of the asks or Petri dishes, with the culture medium above. Centrifuges Autoclave Water Baths: Used to heat cells to their growth temperature. pH Meter: pH variations affect cell metabolism. Freezers and Refrigerators Liquid Nitrogen Containers: Used to preserve cultures in liquid nitrogen. Materials Tubes, syringes, lters. Serological pipettes. Culture Flasks: Their dimensions and shape depend on the type of cell growth and the culture’s purpose (e.g., Western blot to determine protein action requires a large quantity of cells). These asks usually have a lter in the cap for gas exchange, where the air is ltered. Reagents Culture Medium: Provides the nutrients needed for cell growth. Commercial cultures indicate the medium in which they grow. Donated or self-isolated cultures require literature research and possible adjustments. The medium provides nutrients, growth factors, and stabilizes pH and osmotic pressure. Its main components are: Vitamins Phenol Red: A pH indicator – basic pH turns phenol red darker; optimal pH between 7.2 and 7.4 is reddish-orange, at pH 7.0 it is orange, and it becomes progressively lighter as acidity increases. More cells in the culture make the pH more acidic. Bacterial contamination also acidi es the medium by consuming nutrients. For certain studies, phenol red cannot be used due to its estrogenic effect. Glucose Fetal Bovine Serum (FBS): The composition of serum is not fully known and varies between batches, which can in uence cell behavior. Some researchers argue that cells should grow without serum, but many cells under these conditions trigger cell death processes. Antibiotics and Antimycotics: Added to culture media to prevent bacterial and fungal growth. Buffers. Washing Solutions: Typically trypsin, an enzyme that allows the separation of cells from the bottom of asks for further isolation or use in laboratory techniques. CELL CULTURE CONTAMINATION Contaminations can kill cells, affect gene expression, alter morphology, differentiation states, and growth. There are various types of contamination: fl fl fi fi fl fi fl fi fi fi fl  5 Chemical: Occurs through impurities in the medium, buffers, serum, etc., and is the hardest to detect, usually through changes in cell growth patterns. For example, cells that would take 48 hours to double but take longer might indicate chemical contamination. Biological: Can be caused by bacteria, yeast, fungi, mycoplasma, or even by contamination with other cell cultures. Mycoplasma: Mycoplasmas are very small bacteria without a cell wall, often invisible under optical microscopy in cultures. They are only detected when they reach high densities and cause severe damage to the cell culture, altering its behavior and metabolism. Detection requires molecular biology techniques like PCR, which should be done routinely. Cell Culture Contamination: This is a serious problem. A 2010 study estimated that 15-20% of cell cultures being used are not correctly identi ed. Such contamination compromises research results, wastes time and money, and damages the reputation of labs. The solution is to authenticate cell lines using techniques like karyotyping, isoenzyme analysis, STR (Short Tandem Repeats) analysis, etc. These should be done routinely, when establishing a new line, before conducting experiments, and before submitting papers for publication. DETECTION OF CONTAMINATIONS Contamination is mainly detected by observing cultures. Phenol red can indicate contamination through color changes, growth rate alterations, loss of adhesion, and turbidity of the medium. Cell lysis produces fragments similar to the early stages of bacterial contamination, indicating some form of contamination. Vacuolization and the appearance of multinucleated cells are also signs of contamination. SUBCULTURES When maintaining cell cultures that do not have in nite division capacity, subcultures are necessary. Subcultures prolong cell proliferation by transferring cells from a previous culture into a new culture medium rich in nutrients. Through divisions and subcultures, a cell culture develops an in vitro age. This age corresponds to the passage number, i.e., the number of times the cell line has been sub-cultured. The in vitro age is useful for knowing when speci c characteristics change over time in cultures with a nite number of divisions or unstable cultures. CRYOPRESERVATION To preserve cultures and use them later, they need to be frozen. Freezing must be done slowly to reduce the risk of ice crystal formation, which causes cell death, and thawing should be done quickly in a 37°C water bath, resuspending the cells in culture medium. Cryopreservation can be done in two ways: - Congelamento a -80oC: as células duram alguns meses. - Congelamento com Nitrogénio Líquido: este congelamento faz-se em duas fases, uma líquida, a -196oC, e uma gasosa, a -156oC. fi fi fi fi  6 CULTURAS 2D vs CULTURAS 3D Cytology is the microscopic study of isolated cells from tissues, using techniques that facilitate their observation. The cellular samples can be obtained spontaneously or through speci c collection methods, such as exfoliation or aspiration. Below is a detailed explanation of the main types and techniques in cytology: Types of Cytology 1. Exfoliative Cytology: This involves the study of cells that naturally exfoliate from the body, such as those found in body uids (pleural, pericardial, and auricular), in ammatory exudates, urine, and skin mucosa. 2. Aspirative Cytology: This method involves the collection of cells from lesions or organs using a ne needle aspiration (FNA). It is suitable for both palpable and non-palpable masses and helps prevent super cial contamination. Advantages of Cytology Non-invasive. Simple to execute. Fast collection and processing, leading to quick results. Inexpensive techniques. Easy to repeat material collection if necessary. fi fl fi fl fi  7 Disadvantages of Cytology Low cellularity. Low representativeness of lesions. Risk of contamination by blood or bacteria. Provides less information compared to histological samples. Exfoliative Cytology Techniques Imprint In this method, cells are removed by pressing a glass slide against the tissue. This procedure is suitable for: External skin lesions. Exudative lesions. Quick biopsy diagnoses. Intraoperative examinations. Necropsies. Advantages: Good cellular morphology. Rapid detection of microorganisms. Can be combined with other methods. Disadvantages: Low cellularity. Possible contamination. Low lesion representativeness. Scraping Here, the lesion or tissue is scraped with a glass slide or scalpel, and the collected material is spread on a glass slide. It is suitable for the same applications as imprinting, and it can also be used for microbiological culture collection. Advantages: Collects more cells than imprinting. Disadvantages: Worse cellular morphology compared to imprinting. Higher risk of contamination. Low lesion representativeness. Limited to super cial lesions. Swab This technique involves using a sterile swab (cotton swab), moistened with saline solution for dry areas, to collect cells. The material is then transferred to a glass slide. It is suitable for mucous membranes, hard-to-reach areas, ulcers, and microbiological culture collection. Advantages: fi  8 Good morphology and cellular preservation. Higher cellularity compared to previous methods. Disadvantages: Surface contamination. Brush This method involves using a brush, similar to a swab, and is suitable for mucous membranes, such as nasal, buccal, and cervico-vaginal areas. It can be used with an endoscope or bronchoscope. The material collected must be transferred to a glass slide or liquid preservative. Aspirative Cytology This is performed using the ne needle aspiration (FNA) technique, which allows cell collection from internal lesions or organs. It is suitable for: Subcutaneous lesions. Nodular masses. Super cial lesions/palpable masses. Internal organs: The needle can be guided by imaging techniques such as X-rays, ultrasound, and CT scans. Sample Processing Techniques Sample processing depends on the amount of mucus and proteins present. Cytological samples are preserved differently and have different observation times: Low mucus and protein content: For example, urine lasts no more than 2 hours, even when refrigerated. High protein content: Pleural, peritoneal, and pericardial uids last 24 to 48 hours if refrigerated. High mucus content: Sputum and bronchial aspirates last 12 to 24 hours if refrigerated. Immediate processing is preferable, but preservation is necessary when immediate processing isn't possible or if samples need to be sent to external labs. Blood Cytological Processing Blood can be processed using smears or centrifugation, resulting in a "buffy coat smear"—a layer containing most white blood cells and platelets, typically processed in micro-hematocrit tubes with speci c centrifuges. The buffy coat method is used for: Hematocrit percentage. White blood cell concentration. Differential white blood cell count. Cellular morphology. DNA extraction. Hemoparasite diagnosis. Histological processing. fi fi fi fl  9 Electron Microscopy Electron microscopes use electrons instead of photons for imaging. They use electromagnetic lenses in a vacuum for electron propagation instead of optical lenses. Scanning Electron Microscopy (SEM) In SEM, the microscopes are smaller and less complex, and samples are not sectioned to analyze the external surface. The images appear in 3D. Contrast is often made with metal. Transmission Electron Microscopy (TEM) This method allows detailed observation of the cell's interior, and both tissues and isolated cells can be observed. Electrons pass through tissues to produce 2D images. TEM Sample Processing There is no universal protocol for TEM sample preparation, and the process depends on the speci c sample. The main steps include: Sample Collection: Rapid collection is essential. Tissue fragments must be small before xation due to the slow penetration of TEM xatives. Fixation: A critical step in the process. Incorrect xation results in irreversible structure loss. ◦ Temperature: Fixation at 4°C to slow down cell degradation. ◦ Duration, pH, and Osmolarity: Dependent on xatives and tissues. The most common xatives are: Formaldehyde: A nearly universal xative that penetrates well but poorly xes intracellular structures. Glutaraldehyde: Excellent for protein xation but slow in penetrating lipids. Often combined with osmium tetroxide, a lipid xative that also provides contrast. Other stages include dehydration, impregnation, and embedding, typically using resins like Epon, which allows for ultra-thin sectioning (70 nm) suitable for electron microscopy. Cytotoxicity Cytotoxicity assays evaluate the physical or chemical properties of agents that affect cell health and metabolism, targeting mechanisms like: Cell membrane destruction. Inhibition of protein synthesis. Irreversible binding to receptors. Inhibition of DNA synthesis. Different assays are chosen based on detection mechanisms, speci city, sensitivity, and the availability of equipment. Exclusion Assays fi fi fi fi fi fi fi fi fi fi fi  10 These assays use a toxic reagent with a color marker to detect dead cells. The dye only penetrates cells with damaged membranes, coloring them, but some limitations include underestimation of cell death due to delayed membrane breakdown. Advantages: Simple procedure. Requires a small number of cells. Can detect cell death in non-dividing populations. Disadvantages: Not recommended for monolayer cultures. Trypan Blue Assay Trypan blue is a compound that penetrates dead cells and stains them blue. It is simple and inexpensive, but has limitations, such as potential toxicity and dif culty processing large sample numbers. Colorimetric Assays These use biochemical markers to measure cell metabolic activity, using tests like MTT, MTS, XTT, and WST, reading absorbance before and after the compound's action. Advantages: Easy to use and safe. Easily reproducible in replicates. Can be used with equal effectiveness in 2D or 3D cultures. In the WST assay, the colorimetric compound is converted into a water-soluble formazan. Limitations: The MTT process is cytotoxic because the formazan it produces forms crystals and is not water-soluble. fi  11 The incubation time needs to be optimized. Multiple controls are necessary to reduce false positives and negatives. Viable cells may exhibit variations in their reduction capacity due to enzyme regulation, pH, cell cycle, and ion concentration, not just due to toxicity. LDH Assay When exposed to a toxic compound, cells die, and their membranes degrade, releasing LDH into the medium. LDH is reduced by lactase, producing pyruvate and NADH. The H+ interacts with INT, forming INT Formazan, which is also colored. Advantages: Reliable, fast, and easy to evaluate. It is an indicator of irreversible cell death due to membrane damage. Disadvantages: FBS has inherent LDH activity, so its use is often reduced or eliminated from the culture. SRB Assay SRB enters the cell and binds to amino acids, acting as a marker. A toxic compound is then introduced, and protein synthesis is evaluated by the amount of SRB present, which changes the color of the culture and allows measurement of different absorbances.  12 Advantages: Simple, fast, and sensitive. Strong linearity with cell number. Not highly sensitive to environmental uctuations. Independent of intermediate metabolism. Easily reproducible. Disadvantages: In cell aggregates (3D cultures), the assay has signi cantly reduced performance, especially in terms of absorbance. NRU Assay This assay uses the Neutral Red marker, which is water-soluble and accumulates in the lysosomes of viable cells. Advantages: Good marker for lysosomal damage. Quick and easy to evaluate. Disadvantages: While not in uenced by natural factors like temperature and salinity, it is highly affected by pollutants. Fluorometric Assays These assays are applicable to cells in monolayers or suspensions and are more sensitive than colorimetric assays. AlamarBlue (AB) Assay Also known as the resazurin reduction assay, it measures enzymatic activity. Advantages: Inexpensive and more sensitive than tetrazolium (colorimetric) assays. fl fl fi  13 Can be combined with other methods, such as caspase detectors. Disadvantages: Fluorescence interference from test compounds may lead to an underestimation of toxic effects on the cell. CFDA-AM Assay This assay measures membrane integrity through a chemical reaction mediated by esterases, which only function if the membranes are intact. If the membrane is damaged, the marker is not produced. Advantages: Can be used in parallel with the AlamarBlue assay. Disadvantages: Fluorescence interference may occur due to test compounds. GF-AFC Assay This assay measures the level of protease enzymatic activity in viable cells, as this enzyme's activity is a good indicator of cell viability. Advantages: Non-toxic to cultured cells. Suitable for combination with other assays. Luminescence Assays These are the most sensitive assays available. ATP Assay This assay measures cellular ATP levels, as ATP is one of the most sensitive indicators of cell viability. In this assay, there is a linear relationship between luminescent signal intensity and the amount of ATP in the cell. A reaction with the enzyme luciferase produces a uorescent compound. Advantages: fl  14 It is fast, the most sensitive assay, and less prone to generating artifacts compared to other viability assays. The luminescent signal reaches a stable state 10 minutes after the reagent is added. Eliminates the need for handling a culture plate. Disadvantages: High sensitivity is limited by reproducibility. Real-Time Viability Assay In this assay, the pro-substrate and luciferase are directly added to the cell culture medium. Viable cells with active metabolism reduce the pro-substrate into a substrate. This reduced substrate is used by luciferase to produce the luminescent signal. Advantages: Allows real-time measurement of cell viability and cytotoxicity. The rapid decline in luminescent signal after cell death allows for multiplexing. Disadvantages: Pro-substrate depletion by metabolically active cells. The incubation time with the pro-substrate and luciferase must be empirically determined for each cell type and culture density. Genotoxicity Certain situations and factors may not be lethal to the cell but induce damage and alterations in DNA, leading to various consequences. A series of exogenous factors can provoke these changes, as well as endogenous factors. In most cases, DNA damage is enzymatically repaired by the cells; however, this repair has a certain capacity, which can be exceeded. Types of Damage:  15 Methylation: The most common type is methylation of oxygen 6 in guanine. It is very common, highly mutagenic, and only one enzyme can repair it. Breaks. Crosslinks. Oxidation. Others. Consequences: When cells experience genotoxic damage, the rst response is to activate the DNA damage response cascade. Some enzymes in this cascade assess the damage, while others evaluate the quantity of damage, etc. After this evaluation, a decision is made: 1. If the cell can repair the damage, it proceeds with the repair and resolves the DNA damage. 2. If the cell can repair the damage, but the repair is defective and this defect is not detected, genomic instability arises. This is common in carcinogenic agents. 3. If the cell cannot repair the damage, the cell death pathways are activated. Techniques To assess genotoxicity, some of the cellular and molecular biology techniques used include: Western Blot; Comet Assay; ELISA; Immuno uorescence; Chromatography. Comet Assay The Comet Assay is a relatively simple and highly versatile process that measures DNA breaks in animal or plant cells. The more fragmented the DNA, the greater the migration, and consequently, the more extended the "tail" of the "comet" in relation to the nucleus. This DNA migration occurs via electrophoresis. The process was rst introduced by Ostling and Johanson in 1984 under neutral conditions, but later, in 1988, Singh and colleagues introduced alkaline electrophoresis with a pH greater than 13. The Comet Assay is based on the ability of negatively charged DNA fragments to migrate through an agarose gel under an electric eld. The distance of DNA migration depends on the damage: larger DNA strands migrate less than smaller fragments, so the more breaks the DNA has, the smaller the fragments, and consequently, the greater the DNA migration. fi fl fi fi  16 Cell Isolation The cell isolation method for performing the Comet Assay depends on the type of cells being isolated and how they are organized: Cell cultures: In monolayer cultures, cells are generally scraped or trypsinized to detach them and produce a suspension of free cells. However, excessive trypsinization can cause DNA breaks, altering the Comet Assay results. Blood leukocytes: In this case, whole blood can be mixed with agarose, or the leukocytes can be isolated rst. Leukocytes are typically isolated by centrifugation, although excessive centrifugation can cause breaks. Tissue cells: Most animal tissues can be used as long as they can be disaggregated to create suspensions of isolated cells. Plant cells: The nucleus is released by cutting with a knife. The recommended number of cells per gel is around 2x10^4. Cell Embedding Agarose with a low melting point (LMP) is used at 0.5%. The compound is melted in a microwave, avoiding boiling. It is then cooled in a 37°C water bath, where it remains liquid. Next, the required number of cells is centrifuged. After centrifugation, the supernatant is removed, and the pellet is resuspended in about 50 µL by gently tapping. Then, 140 µL of LMP agarose is added to the suspension. Immediately after this addition, 70 µL is pipetted onto a glass slide already covered with normal agarose and topped with a coverslip. The slides are then placed in the refrigerator for at least 5 minutes to allow the gel to solidify. Cell Lysis Lysis is performed using a lysis solution containing a detergent (Triton X-100) and 2.5M NaCl. The detergent acts on the lipids of the cell membrane, causing disaggregation. This disperses the cytoplasm and nucleoplasm, leaving the DNA as a compact structure devoid of histones—the nucleoid. Lysis should be performed at 4°C for at least 1 hour. It can be done overnight, but the longer the gel is prepared, the more likely it is to fail. Alkaline Treatment and Electrophoresis The slides are placed in an alkaline solution within the electrophoresis tank at 4°C and left for 40 minutes. After this treatment, electrophoresis is conducted for 20 minutes at 21V (corresponding to 1V/cm between electrodes). If the alkaline solution depth exceeds 1mm, the maximum current may be exceeded. The excess alkaline solution should be removed. The most common current is 300mA, but voltage is more important. The electrophoresis tank must be level to ensure uniform coverage of all slides. Neutralization and Staining fi  17 After electrophoresis, the slides are placed in a PBS bath for a few minutes. If slides are stored, they should rst be washed with water to remove the buffer and prevent crystal formation. For storage, slides are air-dried overnight. For visualization of comets, either fresh or dried slides must be stained with a selection of dyes, including DAPI, propidium iodide, ethidium bromide, or Sybr. Image Analysis – Visual Scoring In visual scoring, ve DNA damage classes are created based on the size of the comet tail: Class 0: Less than 5%. Class 1: 5-20%. Class 2: 20-40%. Class 3: 40-70%. Class 4: More than 70%. If 100 comets are counted and evaluated, the Total Comet Score (TCS) is calculated as follows: (no. of cells in Class 0 x 0) + (no. of cells in Class 1 x 1) + (no. of cells in Class 2 x 2) + (no. of cells in Class 3 x 3) + (no. of cells in Class 4 x 4). In this case, the TCS will range from 0 to 400. This method does not require software, as comets are selected visually. It requires training, is generally the fastest method, though more subjective, and allows for the evaluation of overlapping comets. Computer-Assisted Image Analysis In this type of analysis, a camera connects the microscope to a computer equipped with image analysis software. In this software, the comet is selected, and the uorescence intensity is measured in different areas of the comet. This method can be: Semi-automatic: The operator manually selects the comets to be analyzed. Automatic: The slide is scanned, and all comets are automatically analyzed. The semi-automatic method requires software but allows for manual selection of comets. It is easy to use and fast if the comet staining is good. This method is more objective, although overlapping comets cannot be evaluated. EVALUATION PARAMETERS fi fi fl  18 Tail DNA percentage: (100 x Tail DNA Intensity) / Total Cellular DNA Intensity. The percentage of DNA in the tail is linearly related to the frequency of DNA breaks. Head DNA percentage: (100 x Head DNA Intensity) / Total Cellular DNA Intensity. Tail length: The distance of DNA migration from the nucleus body, used to assess DNA damage. Tail moment: The product of the tail length and the amount of DNA in the tail. ADVANTAGES OF THE COMET ASSAY Requires a small number of cells per sample. Highly sensitive for detecting DNA damage. Can be used with any type of eukaryotic cell population, both in vivo and in vitro. Non-invasive technique. Quick, producing results within a few hours. DIFFERENT VERSIONS OF THE COMET ASSAY 1. Alkaline Comet Assay This is the previously described method, using a pH higher than 13, to detect DNA breaks, incomplete repair excision sites, and cross-links. 2. Comet Assay with Repair Enzymes Used to detect oxidized purines and pyrimidines. In this method, DNA is incubated with speci c endonucleases targeting certain lesions, such as: ◦ Endonuclease III: Detects oxidized pyrimidines. ◦ Formamidopyrimidine-DNA glycosylase (FPG): Detects purines with open rings and formamidopyrimidines. ◦ T4 UV Endonuclease V: Detects cyclobutane dimers and pyrimidines. ◦ AIKA and AIKD: Detect alkylated bases. ◦ Uracil-DNA glycosylase: Detects uracil erroneously incorporated into DNA. 3. Comet Assay with DNA Repair Detection Measures the repair activity of cellular extracts. Various DNA repair mechanisms are involved in addressing different types of damage: ◦ Single-strand break repair. ◦ Double-strand break repair. ◦ Direct reversal of base damage. ◦ Base excision, removing small base defects and replacing them with new bases. ◦ Nucleotide excision, a similar process to base excision. ◦ Mismatch repair. 4. Not all these mechanisms can be studied using the Comet Assay. Cellular Repair Assay Cells are treated with a damaging agent, incubated with fresh medium, and the repair of lesions is monitored. This method effectively detects damaged bases and breaks using the comet. In Vitro Repair Assay The principle of this technique is that an extract prepared from the cells of interest is incubated with a substrate containing speci c damage. The extract's ability to introduce breaks at the damaged DNA sites (the initial step of repair) is detected using the comet. fi fi  19 COMET-FISH Assay Used to detect damage and repair in speci c sequences or genes. Cells undergo lysis treatment with Triton X-100 and NaCl, leaving highly condensed nucleoids. These nucleoids are incubated with enzymes or genotoxic agents that induce breaks, followed by alkaline electrophoresis (Comet Assay). After the comet assay, the FISH method is applied using probes that recognize speci c genes or sequences, detecting whether they have suffered breaks or not. IMMUNOLOGICAL TECHNIQUES Immunohistochemistry and Immunocytochemistry Immunohistochemistry and immunocytochemistry are two of the most common immunological methods. Their principle is based on the detection of speci c antibodies that bind to a target antigen. This binding is speci c, ensuring reliable results. Antigens can be speci c parts of certain molecules being detected, not just pathogen markers. APPLICATIONS These two techniques can be applied to cells (immunocytochemistry) or tissues (immunohistochemistry). For cells, the technique can be used on smears, cell cultures, or cell suspensions. For tissues, the technique can be applied to whole organs or organisms, cryopreserved tissues, paraf n-embedded, or resin-embedded tissues. ANTIGENS Antigens are molecules with different chemical natures recognized by immune cells. This recognition leads to the production of speci c antibodies by the immune cells. ANTIBODIES Antibodies are proteins produced by activated B lymphocytes after recognizing an antigen. These proteins belong to the immunoglobulin (Ig) group, with IgG and IgM being the most commonly used in immunohistochemistry. Each IgG has a basic structure of four chains—two light and two heavy. Monoclonal and Polyclonal Antibodies Antibodies can be of two types: Monoclonal: Identical antibodies produced by a single clone of plasma cells that react with only one epitope of the antigen. The production of monoclonal antibodies involves creating fi fi fi fi fi fi fi  20 hybridomas, which provide a unique and immortal cell line capable of producing unlimited quantities of highly speci c antibodies. Polyclonal: Antibodies produced by many different cells, reacting with multiple epitopes of the same antigen. Polyclonal antibody production involves activating multiple B lymphocytes, resulting in the production of a large number of antibodies with different af nities for the antigen's epitopes. The antibodies are then puri ed to retain only the desired antigen-speci c ones. CARE FOR GOOD IMMUNOHISTOChemestry Storage and Handling of Antibodies - Follow the instructions of the company manufacturing the product. - Read the data sheets carefully. - Avoid freezing and thawing. - Be careful of contamination from pipetting. - Antibodies should only be kept at room temperature for as long as necessary. Care when carrying out the technique - Fixation should be carried out under optimal conditions and with buffered xatives. - Fixation should not last longer than 48-72 hours to avoid over- xation. - There is no ideal xative for all antigens. - Covered or positively charged slides should be used. - The sections should have adequate time for adhesion to occur and should never dry out. - Each antibody has optimal conditions, so testing them is important and a different protocol should be carried out for each antibody. - Protocols must be optimized for each species. Dilutions with Antibodies As a general rule, the antibody is diluted in a buffer or in diluent antibodies. This dilution is expressed as a dilution ratio. Dilutions with polyclonal antibodies range from 1:100 to 1:2000 or more. With monoclonal antibodies, dilutions are lower, ranging from 1:10 (1 part antibody to 9 parts buffer) to 1:1000. The most suitable dilution is the one that creates the least background. ICQ and IHQ on Paraf n Sections In order to carry out this type of immunological technique on paraf n- xed samples, it is necessary to rst wash with phosphate buffer, then incubate with Streptavidin or an enzyme complex, wash again with phosphate buffer and nally develop and contrast. fi fi fi fi fi fi fi fi fi fi fi fi  21 IMMUNOHISTOCHEMISTRY METHODS DIRECT METHOD In the direct method, only one primary antibody is applied. The primary antibody is against the antigen and has a reporter molecule. This method has advantages and disadvantages. Advantages: - The protocols are shorter and only require one step for labeling. - In cases where multiple antibodies appear in the same species, for example two monoclonal mice, direct labeling may be necessary. - Non-speci c binding is reduced through the use of conjugated primary antibodies. Disadvantages: - The signal is lower. - The cost of antibodies is higher. - Less exibility and fewer conjugates available. Incubation with Primary Antibodies First, an appropriate dilution of the antibodies is made. Then determine the incubation time and temperature (this may be described on the commercial product) and incubate in a humid chamber. It is important to pay attention to the volume of antibodies per section to be incubated and to have different sections for each sample to be tested. It is also very important to have controls: one positive and one negative. STREPTAVIDIN-BIOTIN METHOD This method is highly sensitive and has great signal ampli cation because large amounts of biotin bind to the antibody. The biotin complexes bind to each other and allow the detection of very small amounts of antigen. However, some organs, such as the liver, kidneys and mammary glands, have large amounts of endogenous biotin and may need to be blocked. POLYMER METHOD This technology is very recent and very useful for tissues with a lot of endogenous biotin. It uses a dextran macromolecule to which various antibodies and enzymes bind, greatly amplifying the signal. This technique can be applied to primary or secondary methods. MARKERS FOR IMMUNOREACTIONS To mark immunoreactions, the following can be used - Enzymes: peroxidase (HRP) and alkaline phosphatase. - Fluorochromes: uorescein isothiocyanate (FITC) and Alexa uoride. - Heavy metals: ferritin and other colloids. fl fi fl fi fl  22 The enzymes used utilize the enzyme-substrate reaction to provide the signal in enzyme-linked immunosorbent assay methods. This reaction transforms colorless chromogens into colored end products and allows permanent preparations to be made, visualization under an optical microscope and visualization of a tissue at the same time as immunolabeling. The most commonly used chromogens for HRP peroxidase: - DAB: is converted into a brown product by the enzyme and is insoluble in water. - AEC: produces a rose/red colored product that is soluble in ethanol. ANTIGEN RESTORATION In formalin- xed tissues, it is necessary to recover the antigens so that immunological techniques can be carried out since, due to xative changes, the epitopes of some antigens are masked. As a general rule, this problem is more serious when working with monoclonal antibodies that recognize a single epitope of an antigen. The chemicals involved in formalin xation create methylene cross-links between the antigens and unrelated proteins, which means that the antibody doesn't recognize the antigen. Recovering the antigen breaks these cross-links, allowing it to bind to the antibody. This process can be done using enzymes (pronase, trypsin, pepsin, proteinase K), heat, or a combination of both. Epitope retrieval by heat This process can be done with different forms of heating - microwave, pressure cooker, bain marie - combined with buffers: citrate pH 6, EDTA pH 8 or 9 and commercial solutions for recovery. The recovery solutions and their pH in uence the recovery of the antigen. Some epitopes recover better with a certain pH or buffer. APPLICATIONS OF IMMUNOHISTOCHEMISTRY AND IMMUNOCYTOCHEMISTRY - Neoplasms (diagnosis, prognosis and therapeutic decisions - marking proliferating cells, estrogen receptors). - Infectious diseases. - Basic and applied research. IMMUNOFLUORESCENCE Immuno uorescence is a powerful technique for visualizing intracellular processes and structures using uorescently-labeled antibodies to detect speci c target antigens in a technique that is very similar in principle to IHQ and ICQ. To perform immuno uorescence, two main components are required: - Speci c Antibodies: they form an immune complex to mark the desired molecules (mostly proteins) on the target cells. - Fluorochromes: these are attached to the antibodies (like the reporter molecules in IHQ) and emit uorescence which is detected during microscopy. This technique can be applied to tissue sections, cells in culture, xed samples or fresh samples and is widely used in research and clinical diagnosis. PRINCIPLES OF FLUORESCENCE Electrons are arranged in energy levels around the nucleus of the atom and each level has a predetermined energy value. When an electron absorbs energy from a photon, it becomes excited and rises to a higher energy level, but its electronic stability decreases. The excited state lasts only a few nanoseconds and when it returns to its normal state, the electron releases energy in the form of heat and photons - uorescence. IMMUNOFLORESCENCE TECHNIQUES In this type of technique, antibodies are chemically conjugated to uorescent dyes - uorophores or uorochromes. Fluorophores are organic, low molecular weight compounds that emit light upon photoexcitation. fl fl fl fi fl fi fi fi fl fl fi fl fi fl fl  23 Fluorescent multiplexing methods can be used, i.e. combining several uorescent markers with antibodies, observing them all at once. The labeled antibodies bind directly or indirectly to the antigen of interest, allowing it to be detected. For this detection, uorescence must be detected using techniques such as ow cytometry, uorescence microscopy, confocal microscopy, etc. Avidin-biotin-FITC complex Immuno uorescence techniques based on avidin or streptavidin are widely used to detect biotinylated biomolecules such as primary and secondary antibodies or DNA probes. The formation of the bond between biotin and avidin is very fast and once formed is not affected by extremes of pH, temperature, organic solvents or other denaturing agents. FLUOROCHROMES Fluorochromes are uorescent dyes optimized for excitation and detection from visible to infrared light. The choice of uorochromes to be used should be in uenced by both the application and the excitation wavelength available, so in order to make a good choice, the characteristics of the f luorochromes must be well known. Fluorochromes have several different excitation peaks corresponding to different wavelengths and the distance between the different peaks is called the stroke shift. Some uorochromes have larger or smaller stroke shifts. When multiplexing, it is necessary to select uorochromes with close peaks and carefully match their stroke shifts. There are other physicochemical properties of uorochromes that determine whether they can be used in certain biological assays. FITC FITC has an excitation/emission peak at 490/520nm, i.e. excited by blue light. It can be associated with different antibodies with the help of a reactive isothiocyanate group. It was one of the rst uorochromes used, has high absorbance, excellent uorescence production and good water solubility. TRITC Another frequently used uorochrome, derived from the rhodamine family. TRITC is excited by green light up to 550nm and emits up to a maximum of 573nm. It also binds to antibodies with a reactive isothiocyanate group. It is cheap, but has low uorescence. Cyanines fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fi  24 Cyanines can be linked to nucleic acids or proteins by reactive groups. These uorescent dyes are the basis for many other later uorochromes with greater uorescence and stability. Changes in the conformation of the protein can lead to changes in its uorescence, but Cy5 is still very useful for enzyme measurements. Alexa-Fluor dyes Alexa-Fluor are a large group of negatively charged, hydrophilic dyes that are used very frequently in uorescence microscopy. Maximum excitation is at 493nm and emission at 519nm. They have similar characteristics to FITC but greater stability, luminosity and decreased pH sensitivity. DNA Staining DAPI is a uorochrome linked to an antibody which, instead of binding to proteins or other antibodies, binds to DNA. This can be important for counter-staining samples and localizing the nucleus, as well as for studying nuclear and nucleic acid changes in apoptosis processes. Another uorochrome used for DNA staining is Hoechst, which also stains the nucleus blue if excited with UV light. Another is propidium iodide, which stains the nucleus red, but is impermeable to membranes, so it only stains DNA in dead cells. Hoechst and DAPI stain all cells because they are permeable to membranes. Cross-staining is often done with propidium iodide and Hoechst or DAPI, with dead cells stained red and live cells stained blue after the images have been superimposed. Acridine Orange is another uorochrome used for DNA Staining. The great advantage of this uorochrome is that it changes color depending on whether it binds to dsDNA (green), ssDNA (red), RNA (red) or lysosomes (orange). FACTORS INFLUENCING FLUORESCENCE EMISSION - Concentration of indicator ( uorochrome): if it is too high, the black background that provides contrast is no longer black because too much background is generated. - Extinction coef cient - Ef ciency Quantum of staining - Amount of stained material present in the microscope's eld of view: if the amount is too great, only a blur of light appears, without it being possible to distinguish and individualize structures. FILTERS IN FLUORESCENCE MICROSCOPY The lters used in uorescence microscopy today are highly ef cient, with over 90% re ection of excitation and transmission of emission. These lters attenuate the light emitted during excitation and transmit the light transmitted by the excited specimen to the appropriate receptors. fl fl fi fi fl fl fi fl fl fl fl fi fl fi fl fi fl fl  25 In the case of multiplexing, the primary antibodies must be derived from different species in order to identify the different immune complexes. The uorescently-labeled secondary antibodies then bind to these complexes and must differ enough in their emitted spectrum to allow them to be distinguished under the microscope. IMMUNOFLUORESCENCE CONTROLS In order to correctly interpret microscopy images, it is necessary to have appropriate controls: - Fixed, permeabilized and blocked sample: shows the auto uorescence of the cell compartments. - Sample xed, permeabilized, blocked and with secondary antibodies: reveals non-speci c binding of the secondary antibody. - Negative control: the antigen is blocked by the rst antibody, which prevents the antibody from binding to the speci c epitope. This is to check whether the primary antibody is speci c or not. - Control with samples that are known to express the epitope or not: this allows you to have a positive control or a second “negative” control. - Multiplexing control: the target structure is stained with the different uorochromes in different preparations and then compared. APPLICATIONS This technique can be applied to cells in explanation, cells in culture, tissues and combined with tissue microarrays. LIMITATIONS AND PROBLEMS Microscopes are expensive and observation has to be done in the dark. The permeabilization and xation process can alter cellular architecture and create artefacts that are interpreted as false positives. Each control has to be prepared individually and for each assay, making the technique very expensive in terms of time. Auto uorescence The auto uorescence of samples can cause problems. It can be natural or induced by aldehyde xation. The problems caused by this phenomenon can be minimized by selecting suitable probes and optical lters. Photobleaching Photobleaching is a process in which there is photochemical destruction of the uorophore or uorochrome due to the production of free oxygen radicals as a product of uorescence excitation. It can be minimized by decreasing the intensity and duration of the excitation light, decreasing the availability of singlet oxygen by using uorochromes with a high ef ciency or adding antifade reagents. Fluorescence overlap Fluorescence overlap is a common problem when multiplexing and uorescent signals overlap, such as when using FITC combined with phycoethrin. It is necessary to remove the signal causing the overlap. fi fi fl fl fl fi fi fi fl fi fl fl fi fl fl fl fl fi fi  26 WESTERN BLOT The principle of this technique is the separation of proteins by molecular weight using SDS-PAGE gel electrophoresis. To carry out this technique, it is necessary to destroy the tissues, because we want to quantify the proteins, not locate their structure and relative position in the tissues and cells. The proteins are then transferred to a membrane which is incubated with antibodies speci c to the protein of interest. The speci city of the antibody-antigen (protein) bond guarantees identi cation. SAMPLE PREPARATION This is the rst stage of the technique. The samples can be cellular or tissue-based, but in either case it is necessary to release the cytoplasmic and nuclear content through cell lysis. Various buffers are used to create this lysis and then the proteins are centrifuged to mechanically separate them from the rest of the pellet. It is then necessary to quantify all the proteins using colorimetric assays such as: - Bradford assay (it is limited by incompetence with some detergents used in lysis). - DC Protein assay. - BCA assay. Protein quanti cation is carried out using a dilution curve. After quanti cation, it is necessary to denature the proteins so that their complex three-dimensional conformation becomes linear, so that they can run on the electrophoresis gel. Protein denaturation In order for proteins to run on the agarose gel, they have to be denatured, since their three-dimensional structure does not allow them to run on the gel. Denaturation involves fi fi fi fi fi fi  27 disrupting the secondary, tertiary and quaternary structures of the protein, leaving only the amino acid sequence, and is done by heating at 100°C for 5 minutes in a buffer of SDS and beta-mercaptoethanol. SDS-PAGE The gel is prepared like agarose, with the combs creating wells, but vertically rather than horizontally. To separate proteins of high molecular weight, a lower percentage of polyacrylamide is used in the gel. For low molecular weight proteins, the concentration of polyacrylamide needs to be higher. When loading the proteins onto the gel, it is necessary to load the loading well, which serves as a scale, as in PCR electrophoresis on agarose. The same amount of protein must be loaded into all the electrophoresis wells so that the molecular weight is the only difference in terms of distance traveled on the gel. This is why it is necessary to quantify the proteins at the beginning, so that the amount of protein to be loaded can be de ned. The SDS-PAGE gel has two components: - Stacking gel: this is the upper part of the gel, with a lower concentration of acrylamide to separate higher molecular weight proteins. - Running gel: this is the lower part of the gel, with a higher concentration of acrylamide to separate proteins of lower molecular weight. ELECTRO-TRANSFER A membrane is placed in the gel, onto which the proteins will be transferred. This membrane is made of PVDF and the proteins are transferred using a semi-wet method. The transfer is carried out using an electric current. fi  28 BLOCKING After transfer, all the proteins are on the membrane, even those that are not of interest. The membrane is blocked by incubating it with non-speci c proteins such as albumin and milk proteins. Blocking is done so that no non-speci c binding occurs between the antibodies and the proteins, avoiding the formation of a non-speci c signal. INCUBATION WITH ANTIBODIES The membrane is incubated with the primary antibody, which recognizes the antigen and binds. The membrane is then incubated with a secondary antibody that contains an enzyme, often peroxidase with luminol. The peroxidase acts on the luminol and oxygen peroxide and this leads to chemiluminescence, i.e. the production of light through a chemical reaction, which allows detection. Chemiluminescence is very sensitive and allows samples to be exposed several times, washing to remove the antibodies and repeating the process. DETECTION AND ANALYSIS After incubation with antibodies, the membrane is viewed and the bars and their positions assessed to determine whether the protein is correct, which is done by molecular weight, using beta-actin as a reference. Beta-actin is a protein that forms part of the cytoskeleton and is always very constant (in terms of molecular weight), so it can be used as a control, provided that the treatments under study do not alter this protein, in which case another control will have to be selected. Then, depending on the intensity of the enzymatic signal, it is possible to measure the levels of protein expression. fi fi fi  29 Advantages of Chemiluminescence Detection - Allows multiple exposures to obtain the best image. - The detection reagents can be removed and the entire assay can be re-examined for another protein or to optimize the detection of the rst. - It has a wide-range linear response which allows the detection of large amounts of protein. - It has the highest sensitivity of any other detection method. WESTERN BLOT PROBLEMS - Too strong a signal: the bands stick together and the program can't quantify them. I can reduce the amount of protein loaded or reduce the amount of antibody loaded, reduce its incubation time, change the incubation temperature. - Little or no signal: this can occur due to a mistake in the electro-transfer in terms of layout, excessive time in the transfer. Buffers can cause problems, the voltage in the electrophoresis is wrong, etc. - Too large a background: this often means that the blocking has been done badly. It is necessary to block with a more suitable agent, increase or decrease exposure times, etc. - Stains on the background: the membranes may be slightly damp, the handling of the membranes may have been poor, the use of the same gloves throughout the process may cause contamination, etc. - Non-speci c bands: can be caused by too high a concentration of antibodies, low concentration of blocking agents, etc. - Protein levels change between samples: different amounts of protein may have been loaded into each assay or the control protein may not be valid under the assay conditions. FLOW CYTOMETRY Flow cytometry is a process of cell analysis in ow. It can process thousands of cells per second and analyze up to 50 parameters in a single cell. It also has the ability to identify cell subpopulations, perform a phenotypic analysis of cells, both intracellular and extracellular, perform counting, cell cycle analysis, determine apoptosis, calcium ow, cell division, etc. DEVICE DESIGN The cells undergo hydrodynamic focusing, i.e. they are arranged so that the laser intercepts each cell individually, giving us data isolated from each cell. The interaction of light with the cell in ow cytometry usually occurs by scattering: a particle (in this case, the cell) absorbs the light and emits it in a different direction. It is essentially a scattering phenomenon. SCATTERING Forward Scatter When the cells are intercepted by the laser, a light scattering cone is formed in the direction of laser incidence, with angles between 0.5o and 5o. Depending on the size of the scattering cone, we can get an idea of the size of the cell. The larger the cone, the larger the cell. Side Scatter This dispersion will be proportional to the internal complexity and granularity of the cell. The light in this scatter makes angles of 15o to 105o. DATA ANALYSIS Voltage Pulse It can be quanti ed in terms of its height, width or voltage. The voltage peak is maximum when the cell is completely illuminated by the laser. Flow cytometer data usually appears as a histogram relating the number of cells (yy) and the intensity of the pulse (xx). This histogram generally follows a bimodal distribution. fi fi fi fl fl fl  30 Biparametric scatter plot When the histogram doesn't give enough information, a biparametric scatter plot comparing size and granularity is made. FLUORESCENCE There are various molecules we can use to mark with uorescence, such as propidium iodide, which binds to DNA, or CFSE, which binds to cell proteins without speci city. Antibodies can also be used to make highly speci c bindings to a particular molecule in the cell. These antibodies can be coupled to uorescence-emitting molecules of different colors. In the cytometer there are optical devices that will allow different wavelengths to be directed to the various detectors. These lters are mirrors that can be - - Longpass: wavelengths longer than x nm. - Shortpass: wavelengths shorter than x nm. - Bandpass: wavelengths between x nm and y nm. Color compensation The use of several uorescent markers can cause colors to overlap in the light signals. Color compensation is therefore required. Color compensation is a mathematical method used to solve this problem. fl fl fi fi fl fi  31 APOPTOSIS ASSESSMENT In order to assess apoptosis, it is necessary to use two uorescent markers: propidium iodide and annexin V. This assessment is based on the differences in membrane integrity between viable and dead cells. In the case of propidium iodide, it binds to dsDNA and can only enter dead cells - with compromised membrane integrity (something that happens in late apoptosis). Annexin V binds to phosphatidyl-serine. Phosphatidyl-serine is on the inside of the cell membrane and, when the apoptosis process begins, it moves to the outside of the membrane. It is therefore possible to distinguish viable cells from cells undergoing apoptosis (annexin V uorescence) and cells with delayed apoptosis (annexin V + propidium iodide) in a table like the one below. CELL PROLIFERATION The cells have to be xed, permeabilized and stained with propidium iodide to determine their proliferative state. The cells will be dead due to xation and permeabilization and the propidium iodide (can be CFSE) will bind to their DNA. In this way, they will be distinguished as follows: - G1 phase: they will have less uorescence because they have a smaller amount of DNA. - S phase: they will have intermediate uorescence because they have an amount of DNA between G1 and G2. - M and G2 phases: they will have greater uorescence because they have a greater amount of DNA. Let's get a histogram with the number of cells (yy) as a function of uorescence (xx) with three peaks (one per phase). fl fi fl fl fi fl fl fl  32 PHENOTYPING To phenotype blood cells, for example leukocytes (or even lymphocytes), they need to be incubated with antibodies against the proteins CD3, CD4, CD8, CD19, CD25, CD14 and CD33. These proteins are speci c to the type of leukocyte to be identi ed and therefore, depending on the uorescence emitted, their presence or absence can be identi ed. CELL SEPARATION When passing through the laser beam, the photomultiplier tube sends information about the forward scatter and side scatter of the cells to the computer. According to size (forward scatter) and complexity (side scatter) the cells are marked with a positive, negative or unmarked charge. Upon exiting the ow, the cells are separated by electric current according to their marking. fl fl fi fi fi  33 FLUORESCENCE AND CONFOCAL MICROSCOPY FLUORESCENCE AND FLUOROCHROMES A photosensitive compound absorbs the energy of photons of light of a certain wavelength and passes from the ground state to the excited state. Here, this compound - uorochrome - loses energy returning to the ground state and emits this energy in the form of a photon with a longer wavelength and less energy than the excitation photon. This causes the so-called stroke shifts, which explain the two spectra, an absorption/excitation spectrum, which is more energetic and has a shorter wavelength, and an emission spectrum, which has less energy and a longer wavelength. This is very important when we work with more than one uorochrome. There are various techniques for uorescently marking samples: - Fluorescent dyes: they are absorbed into speci c cell cavities and act as functional indicators. They are used on live cells or permeabilized xed cells. - Immuno uorescence: antibodies are used with the associated uorochrome and it will recognize a region of the cell and bind. They are used on live cells or permeabilized xed cells. - Fluorescent proteins: these proteins modify the cells so that they produce their own uorescent molecules and it is possible to see the movements of the proteins and their interactions. This method can only be used on live samples. APPLICATIONS OF FLUORESCENCE MICROSCOPY MORPHOLOGY STUDIES Morphology studies are achieved through the use of optical sections, projections along the Z axis, image galleries, orthogonal planes and 3D visualization of large volumes. COLOCALIZATION STUDIES In colocalization studies, two or more uorescent signals are used, one for each microscopic structure and then the images are combined, each with its speci c structure in a speci c color. This method is used because of the close proximity between the structures. It should be noted that the signal spectrum must be separable and overlaps eliminated. In addition, care must be taken with the creation of saturation artifacts since the method is based on the intensity of each signal. It is also important to remember that colocalization is not an indicator of molecular interaction. fl fl fl fi fi fl fi fl fl fi fi fl  34 LIVE CELL IMAGING Time-lapse imaging In this technique, many photographs are taken of the preparation over a period of time, creating a time-lapse. The sample needs to be in optimal conditions of temperature, osmolarity, pH and humidity. It is also necessary to avoid phototoxicity by: - Using low-intensity light. - Short periods of light exposure. - Use low resolution and magni cation. - Use a low concentration of uorophores. - Use markers closer to red in the spectrum, if possible. Fluorescence Recovery After Photobleaching (FRAP) FRAP is used when the protein of interest to be observed is moving inside the cell and we want to know how fast it is moving. This method measures the 2D or 3D dynamics of the molecular mobility of proteins (or other molecules) labeled with uorophores in living cell membranes. The process begins by using a confocal microscope and leaching the uorescence from a speci c location, the ROI, with a confocal laser. The return of the uorescent signal is then measured as an indicator of the molecules that have moved in that location. To perform this method correctly, it is necessary to perform it on multiple ROIs and to make corrections for the light signal from leaching and the background. Forster Resonance Energy Transfer (FRET) FRET is used when the protein of interest is interacting with something speci c inside the cell and we want to know where that interaction is taking place. The method itself is the transfer of energy in the form of photons from an excited uorophore - the donor - to another uorophore - the receptor - when they are in close proximity (1-10nm). The effectiveness of this transfer depends on the proximity of the two uorophores. For two uorophores to be used, they must have different excitation and emission spectra and the excitation spectrum of the recipient must overlap with the emission spectrum of the donor. FRET can thus be used to ask for molecular distances, determine molecular interactions between protein partners and ascertain structural or conformational changes within a molecule. CONFOCAL MICROSCOPY The image only shows the part of the sample that is in the focal plane of the microscope. The device has a structure - the pinhole - which only allows the light rays emitting from the focal plane of the fl fl fi fl fl fl fl fl fl fi fi  35 sample to pass through, improving the sharpness of the image and allowing the different uorescences in the sample to be discriminated much better. HEMATOLOGY ANTICOAGULANTS - EDTA: used in all mammals. Preserves blood for 24 to 48 hours. - Heparin: used for reptiles and birds. - Sodium citrate: to isolate platelets and clotting factors. For sh, anticoagulants vary greatly. For some species EDTA is recommended, for others heparin and for still others, such as many elasmobranchs, a mixture of EDTA and heparin. BLOOD COLLECTION - Teleosts: using a lateral approach, blood is taken from the venous plexus near the lateral spine. column. - Elasmobranchs: the caudal venous plexus is generally used via a ventral approach. You can also use an intracardiac puncture or a venous plexus in the dorsal n. - Sea turtles: you can use the jugular vein, the occipital plexus, although this is dangerous, you can use the caudal vein, which allows for smaller volumes, or a sub-carapacial vein. - Dolphins: the venous plexus of the n is often used, as it is highly vascularized. - Sea lion: the large interdigital vessels are used to draw blood. ERYTHROCYTES Just thinking about sh in restrictive terms, we have two large groups that are also divided hematologically: cartilaginous sh and teleost sh. Fish, with the exception of a single family, have nucleated erythrocytes, something that also occurs in birds, reptiles and amphibians. Mammals, on the other hand, have erythrocytes without a nucleus. This means that the lifespan of the cells is much shorter, for example, the erythrocyte of an iguana lives for 3 years and that of a human lives for 120 days. However, anucleated cells maximize blood transport. The erythrocytes of mammals are smaller than the erythrocytes of other animals. As far as sh are concerned, elasmobranchs have larger erythrocytes (elliptical) than teleosts (spherical), since cartilaginous sh have a heart with less contractile capacity, which means they need to increase the size of their erythrocytes to transport more hemoglobin. HEMOGRAM In English, this is called a Complete Blood Count (CBC). The CBC determines the hematocrit (PCV), hemoglobin levels, erythrocyte indices, total erythrocyte, leukocyte and platelet counts, the leukocyte differential and morphology assessment. These processes therefore represent a quantitative and a qualitative side. The CBC is required for - Veri cation of a clinical diagnosis; - Investigation; - Etc. CBC results can sometimes be unspeci c and therefore disappointing. In the case of hemoparasites, the diagnosis is usually suf cient, but they usually serve as a diagnostic aid. It is therefore important to carry out serial blood tests to compare the values obtained. ABSOLUTE NUMBER OF CELLS - Manual methods: Neubauer chamber. Gives very high variability, sometimes in the 20%. - Automatic methods: done with mammalian blood and more accurate. fl fi fi fi fi fi fi fi fi fi fi fi  36 - Quantitative analysis of the buffy coat: this is done using equipment such as the QBC Vet Autoread, which analyzes the uorescence inside the buffy coat. In the buffy coat, platelets are separated (closer to the plasma), monocytes and lymphocytes in the middle and granulocytes closer to the hematocytes. These cells have different uorescences and can be quanti ed. - Impedance counters (Coulter type): these are devices with 1 or 2 aspirators. The blood is aspirated and all the particles are counted in a highly conductive solute through which an electric current passes. Each time a particle crosses the electric current, it emits a recorded pulse. Depending on the size of the particle, the device can slightly differentiate between cells. - Flow cytometry. - New methods: Point-of-care, CellaVision (arti cial intelligence). RELATIVE NUMBER OF CELLS The relative number of cells is obtained from the hematocrit (Hct). It can be determined by automatic methods or by manual methods: the microhematocrit (PCV). Microhematocrit The blood is loaded into microhematocrit tubes and centrifuged in order to separate the blood components: - Plasma: 54% (C). - Buffy coat: 1% (B). - Red blood cells: 45% (A). The packed cell volume (PCV) is given by the following formula: HEMOGLOBIN CONCENTRATION di emoglobina Another piece of information provided by the CBC, the hemoglobin concentration is expressed in g/ dl and determined by photometry in the reaction of the lysate with ferrocyanide, i.e. the cyanomethemoglobin is detected. The concentration value varies in proportion to the number of erythrocytes in the blood and is about 3x higher than Hct in mammals. Abnormal colorations in the plasma may be indications of lipemia or other interferences in this concentration. fl fl fi fi  37 MEAN CORPUSCULAR VOLUME The mean corpuscular volume is expressed in fentoliters and can be determined by analytical methods or by the following formula (where RBC are red blood cells): The value of the mean corpuscular volume can increase with certain conditions such as anemia or vitamin B12 de ciency, or it can decrease, for example, due to an iron de ciency. MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION This measurement corresponds to the average concentration of hemoglobin per erythrocyte and is expressed in g/dl. It is obtained using the following formula: MCHC can increase due to the presence of artifacts or spherocytes and decrease due to anemia. DNA FOUNDING MOMENTS OF MOLECULAR BIOLOGY - Structure of DNA by Watson and Crick. - Introduction of PCR. - Next Generation Sequencing. - CRISPR technology: allows DNA to be modi ed. In eukaryotic cells, a gene can be removed and introduced. NUCLEIC ACIDS DNA and RNA are long polymer chains of small chemical compounds called nucleotides. Guanine and cytosine are nucleotides that are linked by a triple hydrogen bond and adenine and thymine only have a double bond. fi fi fi  38 A gene is a segment of DNA and is the basic unit of heredity. They are located in unique places in the genome - locus. Alleles are variants of the DNA sequence of a given locus. Eukaryotes have genes with a complex structure, large genomes, introns and exons and low gene density. They also have the capacity for alternative splicing, pseudogenes and repetitive sequences and nested genes. Prokaryotes have simple structures, small genomes, no introns and high gene density. Some genes overlap and genes are called ORFs (open reading frames). RNA has three major groups: messenger, transfer and ribosomal. Smaller classes include “small nuclear RNA; small nucleolar RNA” etc. It is single-stranded, the pyrimidine uracil base replaces thymine and the sugar is ribose. Messenger RNA transcribes structural genes and encodes information for protein synthesis. The 5' end is protected - capped. Synthesis of the poly A tail involves cutting the 3' end and adding adenine residues. The ow of biological information is DNA→RNA→PROTEIN The template RNA for protein synthesis (translation): every 3 nucleotides encode an amino acid, the genetic code and tRNAs are the adapters that carry the aa. PCR HOW DOES A PCR CYCLE WORK? DENATURATION This is the rst stage of the PCR reaction and is carried out at 94oC (with slight variations). This high temperature is used to denature the DNA, i.e. to change it from a double strand to a single strand. However, temperature is not the only factor: time is also important. Cytosine and guanine establish a triple bond while adenine and thymine establish a double bond. The triple bond will take longer to denature, so the higher the C-G content of the sample, the longer it will take to denature. ANNEALING This is the second stage of PCR, which takes place at temperatures between 45 and 60oC. This temperature variation is due to the fact that the two primers can have different pairing temperatures. temperatures. The temperature should be as high as possible for both primers, to increase the speci city of binding to the target in the sample. EXTENSION This is the third and nal stage of PCR. It takes place at 72oC, which is the ideal temperature for most thermostable DNA polymerases. This step replicates the target region. The DNA polymerase has no indicator to stop transcription, it only recognizes the binding primer. So it's only from the third cycle onwards, when there are products with the target size, that speci c products start to appear. However, what accumulates in the 1st and 2nd cycle is so minute that it is not detectable. The three-step process is repeated 25 to 40 times to obtain large quantities of copies. After the repeat cycles, the process should be allowed to cool to room temperature, 4oC or -80oC. In theory, 100% ef ciency in the reaction would mean that the DNA product doubles with each cycle, but ef ciency is always lower. KINETICS OF THE PCR REACTION The kinetics of the PCR reaction are divided into 3 phases: - Exponential: corresponds to the situation in which there is a doubling of the product between each