Immunofluorescence and Flow Cytometry PDF

IMMUNOFLUORESCENCE Today, I am delighted to share insights into a vital technique in cellular and molecular biology: Immunofluorescence (IF). This powerful method allows us to visualize specific intracellular processes and structures with remarkable precision. By combining specific antibodies and f...

IMMUNOFLUORESCENCE Today, I am delighted to share insights into a vital technique in cellular and molecular biology: Immunofluorescence (IF). This powerful method allows us to visualize specific intracellular processes and structures with remarkable precision. By combining specific antibodies and fluorescent dyes, immunofluorescence not only enhances our understanding of biological processes but also offers valuable applications in research and clinical diagnostics. 1. The Principles of Fluorescence Let us begin with the basics. Fluorescence occurs when luminescent molecules absorb light at one wavelength and emit it at another. This phenomenon contrasts with phosphorescence, as fluorescence ceases immediately once the light source is removed. In IF, we exploit this property by using fluorochromes to visualize molecular targets. 2. Methods of Immunofluorescence IF operates in two primary modes: Direct Immunofluorescence: This method involves fluorochrome-conjugated primary antibodies directly binding to the antigen. While it is faster and simpler, its sensitivity is limited. Indirect Immunofluorescence: Here, primary antibodies bind to the antigen, and secondary antibodies—linked to fluorochromes—bind to the primary. This amplifies the signal but can introduce challenges like cross-reactivity. Both methods can be applied to fixed or fresh tissues and cells, depending on the experimental needs. 3. Advances in Multiplexing and Fluorochrome Properties A key advantage of IF is its ability to detect multiple targets simultaneously, known as fluorescent multiplexing. This is achieved by using fluorochromes with distinct emission spectra, enabling us to distinguish between multiple molecules in a single assay. The choice of fluorochromes depends on several properties: Excitation and emission wavelengths Quantum yield and photostability Compatibility with the detection system For instance, FITC, a green-emitting dye, and TRITC, emitting red fluorescence, are widely used in tandem. Modern options like Alexa Fluor® dyes provide better brightness and stability. 4. Applications and Visualization Immunofluorescence microscopy has found applications across diverse fields. From studying apoptotic markers in cells to tracking specific proteins in tissues like the mouse cerebellum, it offers a versatile platform. Coupled with advanced imaging techniques such as confocal microscopy, IF allows for higher resolution and the ability to observe dynamic cellular events in 3D. 5. Limitations and Controls No technique is without challenges, and IF is no exception. Some limitations include: Autofluorescence: Cellular components like collagen and lipofuscin can emit background fluorescence, complicating signal detection. Strategies such as optimized filters and antifade reagents help mitigate this. Photobleaching: The photochemical destruction of fluorochromes during imaging can limit signal longevity. Using photostable dyes and minimizing light exposure are effective countermeasures. Proper controls are indispensable for robust IF results. Negative controls, threshold adjustments, and specific antigen blocking are critical for ensuring that fluorescence signals are accurate and free of artifacts. 6. The Future of Immunofluorescence Emerging advancements, such as time-lapse fluorescence microscopy, now enable real- time imaging of cellular processes, expanding the scope of IF. Combined with genetically encoded fluorescent proteins, IF continues to push the boundaries of live cell imaging and molecular research. Conclusion In summary, immunofluorescence stands as a cornerstone of modern biology, merging specificity with visual clarity. While challenges remain, the ongoing development of fluorochromes and imaging technologies promises an even brighter future for this indispensable tool. Flow cytometry is a process of cell analysis in a flow. This analysis is fast, with the ability to process thousands of cells per second and access up to 50 parameters per individual cell. It also can identify cell subpopulations, phenotypic analysis (intra and extracellular), count them, analyze the cycle, determine apoptosis, calcium flow, cell division, cell sorting, etc. Apparatus design The cells are lined up in a single row (hydrodynamic focusing) so the laser can intercede with each cell individually, giving us specific and isolated data from each one. The interaction between cells and light from the laser is through scattering: one cell absorbs light and emits it in a different direction. It is, essentially, a dispersion phenomenon. Scattering Forward scatter: when the cells are hit by the laser light, a dispersion cone is formed in the same direction of incidence of the laser (angles between 0.5º and 5º). Depending on the size of the cone, we can have a rough estimation of the size of the cell. The bigger the cone, the bigger the cell. Side scatter: this dispersion will be proportional to the internal complexity of the cell, as well as its granularity. With this dispersion, the light forms angles between 15º and 105º. Data analysis Voltage pulse Photons emitted by excited fluorophores are routed to a PMT tube (photomultiplier tube). Voltage is applied to PMT making the electrons present for the absorption of light energy from photons. As more photons are detected, more electrons are recruited yielding a greater current on the detector. Sensitivity of the PMT: if PMT voltage is increased the same number of absorbed photons will have a greater current output and PMT sensitivity is increased. Can be quantified by its height, width and voltage. When the cell is completely illuminated by the laser light, it reaches maximum peak voltage (highest point in the graph). Data from flow cytometry is usually represented by a histogram that relates the nº of cells (yy) and the intensity of pulse (xx). This histogram follows a bimodal distribution (normally). Bi-parametric dispersion graph When the histogram doesn't give enough information, we can make a bi-parametric dispersion graph that compares the size and granularity. Fluorescence There are many molecules we can use to mark with fluorescence, like propidium iodide that links to the DNA, or CFSE that links to the proteins of the cell without specificity. We can also use antibodies to make high-specificity links in a determined molecule inside the cell. These antibodies can be joined by molecules that emit fluorescence in different colours. In cytometry, multiple optic devices will divide and send different wavelengths to different detectors. Those filters are mirrors that can be: longpass: wavelenghts above x nm. shortpass: wavelengths below x nm. bandpass: wavelengths between x and y nm. Colour compensation The use of multiple fluorescence markers can create an overlay of colours. So, we need to do some colour compensation. This compensation it's a mathematical method used to solve this issue Evaluation of apoptosis To evaluate this parameter, we need to use 2 fluorescent markers: propidium iodide and annexin V. This evaluation is based on the differences in the membrane integrity of viable cells and dead cells. Propidium iodide links to dsDNA and can only penetrate dead cells - the cell membrane is ruptured (something that happens in late apoptosis). Annexin V links to phosphatidylserine which is found in the inner part of the cell membrane but once apoptosis starts, it is transfered to the outside of the cell membrane. So, it is possible to distinguish viable cells in apoptosis (fluorescence of anexin V) and cells in late apoptosis (anexin V + propidium iodide). Cell proliferation Cells need to be fixed, permeabilized and stained with propidium iodide to determine their proliferation state. This method will kill the cells, so propidium iodide (or CFSE) will link to the DNA and we'll be able to see which stage of the cycle they're on: G1 phase: low level of fluorescence because there is a lower quantity of DNA; S phase: intermediate fluorescence -> quantity of DNA between phases g1 and g2; M and G2 phase: high level of fluorescence because there is a higher quantity of DNA. We can make a histogram with the number of cells (yy) x fluorescence (xx). Phenotyping Example: If we want to phenotype blood cells, eg. leucocytes, we need to incubate them with antibodies against the proteins CD3, CD4, CD8, CD25, CD14 and CD33. These proteins are specific for the type of leucocytes to identify so, depending on their emitted fluorescence, it's possible to identify its presence or absence. Cell sorting As the cells go through the light, the PMT tube sends information on the forward scatter and side scatter of each cell to the computer. According to the size (forward scatter) and complexity (side scatter), cells are branded either with a positive or negative charge or none at all. As they leave the flow, they're separated by an electric current according to their branding. Confocal microscopy Confocal microscopy represents a signi cant leap in imaging technology, o ering high lateral and axial resolution. Unlike traditional microscopy, it employs a laser as its light source and uses a pinhole to block out-of-focus light, thereby enhancing image contrast and detail. This technology enables detailed three-dimensional (3D) visualization of microscopic structures, making it invaluable for morphological studies, live-cell imaging, and protein interactions. The Science of Light and Fluorescence At the heart of confocal microscopy lies the interaction between light and matter. Visible light, an electromagnetic wave, has properties such as wavelength, frequency, and amplitude that dictate its behavior. Fluorescence, a core concept here, occurs when a substance absorbs light of a higher energy (shorter wavelength) and emits light of lower energy (longer wavelength). This phenomenon is elegantly depicted in the Jablonski energy diagram, showing the excitation and emission states. Fluorochromes and Labeling Techniques Fluorochromes are specialized compounds that re-emit light upon excitation. They serve as markers, binding to macromolecules like antibodies, peptides, or nucleic acids. Fluorescent dyes, immunolabeling, and uorescent fusion proteins are some key methods for labeling samples. For example, DAPI stains DNA, while Calcein-AM is used for live-cell imaging. Fluorescent proteins, such as the renowned Green Fluorescent Protein (GFP), derived from Aequorea victoria jelly sh, have revolutionized biological research. Genetic engineering has further expanded the palette of these proteins, o ering various spectral properties for diverse applications. Applications in Biological Research Confocal microscopy nds extensive use in: Morphological Studies: By generating optical sections, researchers can reconstruct 3D images of tissues and cells, exploring their structural intricacies. Live Cell Imaging: This technique reveals dynamic biological processes over time, such as protein movements and interactions. Key techniques like FRAP (Fluorescence Recovery After Photobleaching) and FRET (Förster Resonance Energy Transfer) allow us to study molecular dynamics and interactions with remarkable precision. Protein-Protein Interactions: FRET, in particular, acts as a molecular ruler, measuring distances and structural changes at a nanoscale level. Colocalization Studies: By merging uorescence signals, researchers can identify overlapping structures. However, precautions like eliminating spectral crosstalk are essential to ensure accuracy. Advanced Imaging Techniques fi fi fl fi fl ff ff Confocal microscopy enables a variety of advanced imaging modes: Z-Series and 3D Imaging: Optical sectioning along the Z-axis allows for detailed reconstructions of specimens. Bleaching Techniques: Techniques like FRAP and FLIP (Fluorescence Loss in Photobleaching) help assess molecular mobility and continuity in cellular compartments. TIRF (Total Internal Re ection Fluorescence): Focuses on processes near the cell membrane, enabling detailed surface studies. Challenges and Precautions Despite its versatility, confocal microscopy has limitations. Ensuring optimal environmental conditions, avoiding phototoxicity, and careful selection of uorophores are crucial for reliable results. Moreover, researchers must mitigate artifacts like saturation and spectral overlaps during imaging. Conclusion Confocal microscopy has transformed our ability to visualize and analyze biological systems, o ering unparalleled insights into cellular structures and dynamics. Its applications in marine biology and other scienti c elds continue to expand, paving the way for new discoveries. As we re ne these techniques, the potential for groundbreaking research only grows. ff fl fi fi fi fl  Hematology General Principles in Hematology appropriate techniques and materials is crucial to ensure reliable results. Preserving Blood Samples: Di erent anticoagulants are used depending on the species and the purpose of the study: EDTA is the most commonly used anticoagulant for mammals, preserving blood integrity for up to 48 hours. Heparin is suitable for reptiles and birds, but its application in mammals is limited due to its tendency to cause platelet aggregation and a bluish discoloration in the background. Sodium citrate is primarily used for studying platelet function and conducting coagulation tests. Collection Sites: Blood collection methods vary across species: In mammals, common venipuncture sites include the cephalic, jugular, and saphenous veins. In sh, the caudal vein is frequently accessed, and ne needles are essential to minimize damage to small, delicate species like salmonids or cat sh. Additionally, it's critical to ensure that the volume collected does not exceed 10% of the mammal's body weight or 5% of a sh's body weight. Erythrocyte Characteristics: The morphology and function of erythrocytes can vary signi cantly: Elasmobranchs (e.g., sharks) have larger but fewer erythrocytes compared to teleost sh. Despite this, their packed cell volume (PCV) is lower. Unlike mammals, sh do not rely on bone marrow for hematopoiesis. Instead, blood cell production occurs in the spleen, thymus, anterior kidney, and Leydig’s organ. Understanding Blood Counts The complete blood count (CBC) is one of the most important tools in hematology. It provides both quantitative and qualitative insights into a patient’s health. Components of a CBC: Quantitative parameters include measurements such as hematocrit (PCV), hemoglobin concentration, erythrocyte indices, and total counts of erythrocytes, leukocytes, and platelets. Qualitative analysis focuses on leukocyte di erentiation and the assessment of cell morphology. When Is a Blood Count Necessary? Blood counts are essential for: Investigating systemic diseases. Evaluating abnormal changes in the number or proportion of blood cells. Pre-surgical evaluations. Diagnosing speci c conditions, such as hemoparasite infections. Diagnostic Value: ff fi fi fi fi ff fi fi fi fi  While blood counts may sometimes yield non-speci c results, they can be invaluable in providing a diagnosis or supporting other clinical ndings. In some cases, serial blood counts over time are more insightful than a single measurement. Methods of Blood Analysis Di erent methods are employed to analyze blood, ranging from manual techniques to sophisticated automated systems: Manual Methods: Techniques such as the use of a Neubauer hemocytometer and Natt & Herrick’s stain allow for the calculation of absolute cell numbers. However, these methods are labor- intensive and prone to variability. Automated Techniques: - Flow Cytometry: This advanced method uses lasers to analyze cells based on size, granularity, and internal complexity. It is highly accurate for leukocyte di erentials. - Coulter-Type Impedance: This method measures changes in electrical impedance as cells pass through an aperture. Although e ective for cell counts, it has limitations in di erentiating certain cell types, particularly nucleated erythrocytes. - Point-of-Care Devices: Emerging technologies such as HemoCue devices o er portable solutions but may have variable accuracy depending on the species (e.g., less reliable for dogs). Blood Smear Evaluation Microscopic examination of blood smears provides critical insights into cell morphology and the presence of abnormal or parasitic inclusions. Preparation and Staining: Blood smears are stained using protocols like Di -Quik, which enhances visualization of cell components. The smear is then examined under immersion oil using a high-powered microscope. Systematic Evaluation: A systematic approach ensures that all cell types are evaluated, focusing on both quantity and morphology. Leukocyte assessments include di erential counts (e.g., lymphocytes, neutrophils) and subjective estimations based on elds of view. Challenges in Blood Smear Analysis: Di erentiating certain cell types can be di cult, particularly: Fish: Lymphocytes may be mistaken for thrombocytes. Mammals: Nucleated erythrocytes can resemble lymphocytes. Rare cell types, such as basophils, require careful identi cation due to their infrequency. This detailed overview highlights the diverse approaches and considerations involved in hematological analysis across di erent species. Whether for clinical diagnosis or research purposes, these methods provide a comprehensive understanding of blood physiology and pathology ff ff ff ff fi ff ffi ff ff fi fi fi ff ff  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. - - Quantitative analysis of the buffy coat: this is done using equipment such as the QBC Vet 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: fi fi fi fi fi fl  - Plasma: 54% (C). - Bu y 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. 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. fi ff fi Immunohistochemistry in Marine Sciences At its core, IHC relies on the speci city of antigen-antibody interactions, visualized through markers or reporters under various types of microscopes. Principles of IHC and its Methodology IHC is distinct from related techniques like immunocytochemistry, which deals with isolated cells, and immuno uorescence, which utilizes uorochromes for visualization. IHC is applicable to a wide variety of samples, including cryopreserved, para n- embedded, and resin-embedded tissues, as well as whole organs. The antibodies used in IHC can either be monoclonal—highly speci c and produced by a single clone—or polyclonal, which react with multiple epitopes but may show variability between batches. The preparation of tissues for IHC involves steps such as dewaxing, hydration, antigen retrieval (using enzymatic or heat-based methods), and blocking of endogenous peroxidases or nonspeci c reactions. These processes ensure optimal conditions for antibody-antigen interaction. Key Techniques and Methods IHC can be performed using direct or indirect methods: Direct Method: Involves a primary antibody labeled with a reporter molecule. Indirect Method: Employs a primary antibody against the target antigen and a labeled secondary antibody for detection. This enhances signal ampli cation. Advanced methods like the Labeled Streptavidin-Biotin (LSAB) system and polymer- based approaches further increase sensitivity and reduce background noise, making them suitable for low antigen concentrations or tissues with endogenous biotin Applications of IHC The applications of IHC span various domains: Diagnostic Pathology: Detection of neoplasms, infectious diseases, and prognostic markers like Ki-67, HER-2, and estrogen receptors. Basic and Applied Research: Studying cellular processes and developing therapeutic interventions. Marine Sciences: Although antibodies speci c to sh species are limited, IHC plays a role in identifying and studying biomarkers in aquatic organisms. Optimization for di erent tissues and species remains a challenge, requiring tailored protocols. Technical Considerations and Best Practices For successful IHC, certain critical factors must be maintained: Proper antibody storage, avoiding repeated freeze-thaw cycles. fi fl fi fi fi fi fl fi ffi ff Optimizing xation and antigen retrieval for each antibody and tissue type. Careful selection of antibody dilutions to minimize background staining while ensuring strong signals. Furthermore, tools like Antibodypedia and Biocompare help researchers select suitable antibodies, especially in less studied areas like marine sciences. Conclusion In conclusion, immunohistochemistry stands as a versatile and powerful tool for visualizing molecular processes in cells and tissues. Its applications in diagnostics, therapeutic decision-making, and marine biology underscore its importance. However, as with any sophisticated technique, achieving accurate and reproducible results demands attention to detail and rigorous optimization. Cellular line and culture A cell culture can be created form the cell of a speci c tissue (vegetale o animale) messe in colter o in un medium e quindi in sospensione o in una ask. Lqueste cellule sono estrrmamente eterogenee ma conservano le caratteristiche principali delle cellule fi fi fl  dell’organismo. Quindi simulano meglio le reazioni dell’organismo ad un determinato compound. Poi ci sono le cell line che invece sono colture di cellule che possono essere nte or in nite dipende da quante volte è possibile, l nite sono quelle che hanno un liminte massimo di divisioni cellulari. Solitamente le cellule di queste colture possono avere o delle mutazioni spontanee che le rendono immortali.o possono essere formate con cellule di tumori—> tramite a virus infections. Queste cellule hanno il vantaggio di essere omologhe e molto simili ma dopo una serie di moltiplicazioni potrebbero perdere delle caratteristiche importanti. Crescono molto velocemente ma hanno meno possibilità di essere infettate, danno risultati + riproducibili. Il numero di passaggi quando si inizia un eperimento dovrebbero essere il + basso possibile. Prima di iniziare un esperimento sarebbe meglio identi care la cell line con test appositi e tre una PC per vedere se c’è qualche tipo di organismo che ha contaminato la coltura. Possono crescere o in un monolayer aderenti alla ask or in suspension—> limite in un caso la super cie e nell’altro il volume. Morfologia: - broblastic: monolayer, bi o multipolar, grow attach to the ask - Epithilial: poligonal, attach to the ask - Linphoblastic: spherical—> usually grow in suspensions. Lab conditosin: biosafety: is good no to contain cell—> very easy, and it is important to be careful with the exposure. So there are a lot of practical things to do to avoid continuation of the cells or of the environment. There are 4 biosafety level: - from 1 to 4—> one is the lowest—> open acess to lab, dispositivi di protezione, no laminar ow hood needed. 4—> shower outside, automatic door, personale selezionato, mute, aria dell laminar ow e del personale è diversa. Filtri HEPA. Reagent use for cell colture and cell line: - medium—> is the liquid with the nutrients, vitamine and most of the things that are necessary for the cell, growth factor, stabilizzatore di pH. Phenol red—> is an indicator for the pH changing —> can be a sign for cell cotamination - Fbs: fetal bovin serum—> partially unde ne, sometimes helps with cell grows - Antibacterila e antifungino antivirale —> blocca la crescita di batteri, funghi e virus —> contaminazione biologica - Micoplasma—> bacteria without cell walls very small di cult to detect fi fi fl fi fi fl fl fi fi fl ffi fl fi Genotoxicity test: - Ames Test (Bacterial Reverse Mutation Test): a widely used biological assay that uses bacteria to assess the mutagenic potential of chemical compounds, helping identify substances that may cause genetic alterations. Remains a cornerstone in genotoxicity screening. - Chromosomal Aberration Test: used to identify substances that can cause damage to chromosomes. Identi es structural changes in chromosomes, including breaks, gaps, and rearrangements. - Sister Chromatid Exchange Assay (SCE): Measures the exchange of DNA between sister chromatids as an indicator of genetic damage. - The mouse lymphoma Tk assay (MLA): A popular laboratory test that quanti es genetic alterations a ecting expression of the thymidine kinase (Tk) gene. Detects gene mutations and chromosomal alterations in mammalian cells. - HPRT Gene Mutation Assay: Detects mutations in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene, a key enzyme in the purine salvage pathway. The assay is commonly performed on mammalian cells and helps assess the mutagenic potential of chemical substances. - Micronucleus Assay: A widely used test that detects chromosomal damage by measuring micronuclei, which contain chromosomal fragments or whole chromosomes not included in daughter nuclei during cell division. - Comet Assay (Single-Cell Gel Electrophoresis): Detects DNA strand breaks in individual cells, providing insights into damage and repair mechanisms from environmental and chemical exposures. E ective for evaluating genotoxic agents. CYTOTOXICITY TESTING METHODS - ATP Assay (Adenosine Triphosphate): Quanti es ATP levels, indicating cell viability based on the luminescence generated from ATP reacting with luciferase. - Caspase Activity Assays: Detect and quantify caspase activity in cells, offering insights into apoptosis. - Trypan Blue Exclusion Assay: Uses trypan blue dye to stain dead cells, allowing for counting of viable cells under a microscope. - Eosin Y Exclusion Assay: Comparable to trypan blue, eosin Y stains dead cells to assess viability. - Propidium Iodide (PI) Assay: Utilizes PI to stain dead cells, often used in ow cytometry. - MTT Assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide): A method that measures cell metabolic activity by detecting the conversion of MTT into purple formazan crystals, indicating cell viability and cytotoxicity levels. - XTT Assay: Measures cellular metabolic activity by reducing the yellow tetrazolium salt XTT to a highly colored formazan dye through dehydrogenase enzymes in metabolically ff fi ff fi fl fi  active cells. Easier than MTT as it eliminates the solubilization step, widely used for drug screening. - Sulforhodamine B Assay (SRB): Based on the ability of SRB dye to electrostatically and pH-dependently bind to basic amino acid residues in proteins of trichloroacetic acid- xed cells. Commonly used to assess cytotoxicity and cell proliferation in long-term studies. - Neutral Red Uptake Assay (NRU): Quanti es xenobiotic-induced cytotoxicity by measuring the incorporation of neutral red dye into lysosomes of living cells. Also used for testing toxicity in chemicals and cosmetics. - LDH Assay (Lactate Dehydrogenase): Monitors cell membrane integrity in cytotoxicity assays. LDH is a stable cytoplasmic enzyme present in all cells, rapidly released into the culture upon plasma membrane damage. - Alamar Blue Assay (Resazurin Reduction): Widely utilized to assess cytotoxicity, cell proliferation, and metabolic activity across various elds of toxicology. - Annexin V—> binds to phosphatidil serina—> in apoptotic cell is translated on the outside. - DAPI Staining (4',6-Diamidino-2-Phenylindole): Uses DAPI to stain DNA in live and xed cells, allowing assessment of cell number and viability. Cytotoxicity assays such as MTT, XTT, and Alamar Blue can assess proliferation, but more specialized assays offer greater speci city for measuring cell division fi fi fi fi fi  Comet essay help to undertand in a very precise way if a compound can damage and break the DNA or not. It is sensitive and can calculate the ripair mechanism. There are more them 1 type : - alkaline comet essay is used to detect double and single strand DNA damage, I - Neutral: is speci c for the double strand break - Enzime is used to single nueclatide change and repair—> is extremely speci c. Firs step is to put the cell on a slide in agarose and, to lyse the cell to make the DNA come out of it, then then cam be an alkaline incubation in the rst type and the slide can go trough electrophoresis-_> the DNA that is negatively charge goes to the anode—> if I migrate to the same place—> this means that there are not breaks and the compound is not genotixic—> if there are breaclks is possible to see after the coloration (can be done by uorochrome, the tail of the comet. In marine organsim it is possible to do di erent things: to extract cell from the blood or from form the liver, or it is possible to use zooplancton and toplacoton, after this the viability is counted and the the cell are put on the slides—> and then on a lysis bu er to break the cell—> to prevent osmotic stress during lysis—> there are ione and pH tath mimes the marine one. Slides are rinsed with neutral or alkaline bu er, depending on the type of the assay Marine cells, especially those from sh or invertebrates, may require optimization of the bu er conditions to accommodate their speci c DNA properties/characteristics* and prevent excessive damage during electrophoresis. Western blot: it is used to asses the protein in the semi quantitive analysis of a mixture of proteins. It uses antibody. It is done using SDS Page—> as a gel for elettroforesi, ha lo stesso principio dell’acrilammide, to extract the protein is required.a buffer that usually contains salt, buffer for the pH and reducing agents to avoid protein oxidations. After this it can be done. Protein quanti cation trough a colorimetric assay—> spettrofotometro to understand how many proteins are n every singola sample, Quindi per proteine di peso molecolare elevato è necessario avere meno concentrazione di sms page (inversamente proporzionale) e viceversa. SDS page è importante perchè aiuta a mantenere le proteine nel loro stato lineare i n maniera che possano scorrere attraverso il gel e dona probabilmente anche una carica negativa, the protein are than separated due to molecular weight—> thought a resolving gel—> in which the proteins are actually separated and a stacking layer in which the sample are uploaded. After this the gel is transferred to a membrane in a speci c macchinario that always use the electric eld to do the transfer, it is used electrobloking a speci c proteins to trmake the trasfer. Than the buffer use in this chamber (anodo, catodo, and that I always kept wet with sheets) contains methanol, that helps to disassociate DSD and to stabuikyze the proteins. than the membrane is block with no speci c proteins, the the antibody are add—> direct or indirect method—> primary and secondary antibody—> uorescence/signal—> light/ detector—> quanti cation of the proteins ff fl fi fi fi fi fi ff ff fi fi fi fi fl fi fi ff fi