Stem Cell Biology (BIO414) Lab 1 - Introduction to Cell Culture PDF

Stem Cell Biology (BIO414) Lab 1 Introduction to Cell Culture COURSE COORDINATOR: DR. SHAZA AHMED COURSE ASSISTANT: L.A. AYA EL-SHARKAWY Fall 2024 Cell Culture  Cell culture refers to the removal of cells from an animal or plant a...

Stem Cell Biology (BIO414) Lab 1 Introduction to Cell Culture COURSE COORDINATOR: DR. SHAZA AHMED COURSE ASSISTANT: L.A. AYA EL-SHARKAWY Fall 2024 Cell Culture  Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment.  The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line that has already been established.  Primary Culture Cell Line Primary culture refers to the stage of the After the first subculture, the primary culture after the cells are isolated from the culture becomes known as a cell line or tissue and proliferated under the subclone. Cell lines derived from primary appropriate conditions until they occupy all cultures have a limited life span (i.e., they of the available substrate (i.e., reach are finite), and as they are passaged, cells confluence). At this stage, the cells have to with the highest growth capacity be subcultured (i.e., passaged) by predominate, resulting in a degree of transferring them to a new vessel with fresh genotypic and phenotypic uniformity in the growth medium to provide more room for population. continued growth. Primary vs Immortalized Cells Primary Cells Immortalized Cells Are taken directly from living tissue. For Are either derived from cancerous tissues or cultivation, the original tissue fragment is transfected with oncogenes. Due to their dissociated enzymatically, chemically, or malignant background, they divide mechanically into single cells which can be indefinitely. This fact makes them very easy seeded on culture flasks. Primary cells are to culture as a cell line. They are also robust much harder to cultivate than immortalized and affordable. cell lines. Their survival rate is low and many Commonly used immortalized cell lines are of them don't divide. Moreover, their HEK, A549, Jurkat, MDCK, COS, or Vero cells. genetic manipulation can be challenging. Cell Culture Laboratory  Cell Culture Labs should be at least of Biosafety Level 2 (BSL-2).  Cell Culture Equipment: 1. Cell Culture hood: Class II biosafety cabinet, laminar flow hood. 2. CO2 incubator 3. Water bath 4. Centrifuge 5. Refrigerator and Freezer (4℃, -20℃ and -80℃) 6. Inverted Microscope 7. Autoclave 8. Liquid nitrogen or cryostorage container Cell Culture Supplies 1. Cell culture vessels: flasks and plates. 2. Assorted sterile pipettes and pipetting devices 3. Cryovials 4. Falcons and eppendorfs 5. Controlled-rate freezing container (canister) Following aseptic technique during cell culture 1. Sterile work area: o The cell culture hood should be properly set up and located in an area that is restricted to cell culture and is free from doors, windows and other equipment and has no traffic. o Ultraviolet light should be used to sterilize the air and exposed work area and surfaces in the cell culture hood between uses. o The work surface should be uncluttered and contain only items required for the ongoing experiment. o Before, during and after use: the work surface should be disinfected by wiping with 70% alcohol. Following aseptic technique during cell culture 2. Good Personal Hygiene: o Your hands should be washed before and after working with cell cultures. o Working personnel should wear their personal protective equipment to reduce the probability of contamination from shed skin, dirt and dust from clothes. Following aseptic technique during cell culture 3. Sterile Reagents and media: o Any reagents used, media or solutions prepared in the laboratory should be sterilized prior to use with the appropriate sterilization procedure. Following aseptic technique during cell culture 4. Sterile Handling: o Always wipe your hands and work area with 70% alcohol. o Wipe the outside of the containers, flasks and dishes with 70% alcohol before placing them inside the cell culture hood. o Use sterile glass or disposable plastic pipettes and a pipette aid to work with liquid. o Always cap the bottles and flasks after use and seal multi-well plates with parafilm to prevent microorganisms and airborne contaminants from entry. Stem Cell Biology (BIO414) Lab 2 Media Types for Cell Culture COURSE COORDINATOR: DR. SHAZA AHMED COURSE ASSISTANT: L.A. AYA EL-SHARKAWY Fall 2024 Optimum conditions for cell culture  Cell cultures should be incubated in a sterile incubator with a tightly regulated temperature (e.g., a water-jacketed incubator) and CO2 concentration.  Most cell lines grow at 37°C and 5% CO2 with saturating humidity. Optimum Culture conditions  Culture conditions vary widely for each cell type, but the artificial environment in which the cells are cultured invariably consists of a suitable vessel containing the following: 1. A substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals) 2. Growth factors 3. Hormones 4. Gases (O2, CO2) 5. A regulated physico-chemical environment (pH, osmotic pressure, temperature). Cell Culture Media  There are six main ingredients found in cell culture media: 1. Carbon source (e.g., glucose). 2. Buffering system (e.g., HEPES). 3. pH Indicator (e.g., phenol red). 4. Serum. 5. Metabolites, vitamins, and minerals. 6. Antibiotics. 1. Carbon Source  This is the energy source for the cells. Every growth media requires a carbon source that cells can metabolize. The carbon source could be glucose, or any other sugars such as galactose, hexose, fructose, or other carbon sources (e.g., pyruvate or glutamine). 2. Buffering System  Cells require a very specific pH from 7.2 to 7.4. If the pH moves out of this narrow range, cells cannot perform their usual cellular functions optimally to grow and survive.  In incubators with high carbon dioxide levels, CO2 reacts with water to produce hydrogen ions that turn the media acidic.  To counteract the acidity generated by CO2 and cellular byproducts and maintain the optimal pH in culture, most media types use a chemical buffering system.  The most common buffer system for mammalian cells with minimal biological impact is sodium bicarbonate. Sodium bicarbonate reacts with the hydrogen ions generated by CO2 and sequesters them to maintain pH. 3. Phenol Red  Because maintaining the correct pH is so critical to cell culture, many media formulations include a pH indicator called phenol red that turns yellow in acidic conditions (pH < 6.8) and pink in basic conditions (pH > 8.2).  Media without phenol red is also available for colorimetric assays, such as the MTT proliferation assay, and for fluorescence experiments where the color spectrum of phenol would likely interfere with the readouts or image acquisition. 4. Serum  An essential component for many culture systems is serum, commonly fetal bovine serum (FBS). Derived from blood, serum is an undefined mixture of sugar, salts, lipids and growth factors.  Serum should be heat inactivated before use in culture, as it should be heated at 56°C for 30 minutes to inactivate the complement system (a group of proteins present in sera that are part of the immune response) as well as to destroy mycoplasma contaminants. Role of Serum  Provide essential nutrients; various amino acids, vitamins, inorganic minerals, fat, and nucleic acid derivatives, which are essential for cell growth.  Provide adherence as it contains some components; fibronection, laminin, etc., which can promote cell adherence.  Provide hormone and various growth factors; fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and others.  Provide binding protein(s): the albumin carries vitamins, fat (fatty acid, cholesterol) and hormones. Transferrin carries iron.  Provide protection for some specific cells: Some cells (such as epithelial cells, myeloid cells) can release protease, which can be neutralized by the anti-protease ingredient in the serum. Serum is widely used to terminate the effect of the trypsin. Serum albumin facilitates the serum viscosity and protects the cell from mechanical damage, especially in the suspension cell culture. The trace elements and ions play very important role in metabolic detoxification. 5. Metabolites, Vitamins and Minerals  Amino acids; ex.: L-glutamine which is an energy source and plays an important role in cell metabolism process.  Vitamins/cofactors: act as coenzymes in cell metabolism processes.  Inorganic salts; calcium and magnesium: help in cell growth. 6. Antibiotics  It is advised to include antibiotics in cell cultures to prevent unwanted bacterial or fungal growth, even when practicing aseptic technique. The most popular choice of antibacterial supplement is a combination of penicillin and streptomycin (pen/strep) at a final concentration of 50–100 IU/mL penicillin and 50-100 µg/mL streptomycin in complete cell culture (1% of complete media).  Disadvantages of using antibiotics: 1. They have unwanted side effects on cells, including significantly altering gene expression. 2. Can mask low levels of bacterial contamination. 3. Acquiring of resistant bacterial strain due to heavy use of antibiotics. 7. Additional Supplements  Hormones are very important to maintain cell function and status (differentiated or undifferentiated).  Some hormones have promoting growth effects on different cell types. For instance, insulin can promote the use of glucose and amino acids in the cell. Some hormones are cell-type specific, as hydrocortisone that can promote the growth of epidermal cells and prolactin that induces the proliferation of mammary epithelial cell. Basal vs Complete Media Basal Media Complete Media Typically consists of a minimal nutrient Provide a wider range of nutrients, solution, providing only the essentials including additional amino acids or required for growth, such as carbon vitamins. Complex media are often used source, amino acids, and salts. Basal when the goal is to obtain a large media are often used for the selective quantity of cells. Complex media can growth of specific cells, as they allow for also contain additional components, the addition of specific nutrients or other such as blood, serum, or tissue extracts, factors required for growth. which can provide additional growth factors and support the growth of more fastidious cells. Media Features A minimal medium, first developed in 1959. Useful as a base for modification for specific requirements. EMEM (Eagle's minimal essential medium) It only contains 12 kinds of non-essential amino acids, glutamine, 8 vitamins and some basic Inorganic salts. Similar to EMEM but contains approximately four times as much of the vitamins and amino acids present in the original formula and two to four times as much glucose. DMEM (Dulbecco's Modified Eagle's Additionally, it contains iron and phenol red. Medium) DMEM is suitable for some tumor cells with faster growth speed and difficult attachment; epithelial and fibroblast cells. RPMI-1640 (Roswell Park Memorial A more complex medium commonly used for human lymphocytes. Can support a wide Institute -1640) range of cell types; tumor cells, normal cells, and primary culture cells. A modified version of DMEM. It is well suited for difficult proliferating, low-density cell IMDM (Iscove′s Modified Dulbecco′s cultures, including hybrid cell selection after cell fusion, selection of transformed cell Medium) after DNA transfection. A modification of Basal Medium 5A. Used in the culture of many primary cells and explants from biopsies; as it supports the growth of primary cultures derived from McCoy's 5A adrenal glands, bone marrow (normal), gingiva, lung, mouse kidney, skin, spleen and other tissues. It is a general-purpose medium for primary and established cell lines. Commonly used for serum-free growth of Chinese Hamster Ovary (CHO) cells. Also used Ham's F-12 for serum-free growth of other mammalian cells Cell Culture Contamination  Cell culture contaminants can be divided into two main categories; 1. Chemical contaminants such as impurities in media, sera, and water, endotoxins, plasticizers, and detergents. 2. Biological contaminants such as bacteria, molds, yeasts, viruses, mycoplasma, as well as cross contamination by other cell lines. While it is impossible to eliminate contamination entirely, it is possible to reduce its frequency and seriousness by gaining a thorough understanding of their sources and by following good aseptic technique. 1. Bacterial Contamination  Many bacterial strains can grow in commonly used media in mammalian cell culture. Due to rapid logarithmic growth, the signs of contamination are easily detectable within a few days of the initial contamination.  Loss of medium clarity.  Faster acidification of medium (medium turns orange/yellow if phenol red is present).  A thin bacterial film appears on the surface of cell culture vessels/flasks.  Bacteria can be identified by a brightfield microscope check (20–60x magnification) as slowly moving, dark, round- or rod-shaped objects that are significantly smaller than mammalian cells. 2. Fungal; Yeast/Mold Contamination  Similar to bacterial contamination, yeasts can grow rapidly in mammalian culture media, leading to a loss of medium clarity, but at a slower rate.  Relatively rapid progression from initial infection to a serious threat.  pH is unchanged or slightly increases at later stages of the contamination.  Brightfield microscope check (20–60x magnification): rod-shaped or branched filamentous objects that become visible to the naked eye at later stages of contamination.  Many fungi can produce spores capable of surviving for a long time in a dormant state in hostile environments and, therefore, in the event of fungal contamination it is particularly important to decontaminate all working surfaces and incubators to prevent repeated contaminations. 3. Viral Contamination  Viral contaminations are not very common but can potentially pose a serious health threat to cell culture personnel.  Viruses are hard to detect because they do not affect cell culture growth and cannot be seen with a brightfield microscope.  Dedicated assays (e.g., ELISA, PCR) can be used to test for infections. 4. Mycoplasma Contamination  Mycoplasma are very small (0.2um) bacteria characterized by their lack of a cell wall. Unlike standard bacterial contamination, mycoplasma often grow at a slow rate in infected cultures and can be present with no visible signs of infection such as loss of clarity or change of pH.  Mycoplasma do not contain cell walls, so they cannot be seen with a standard brightfield microscope.  Due to their slow growth rate, mycoplasma infection may develop for a long time without any signs.  They do not usually kill the mammalian cells they infect, but significantly impact cultures by altering cellular metabolism, causing chromosomal aberrations, slowing cell growth, and interfering with cell attachment. In short, they are likely to dramatically influence the results of most experiments performed using affected cell lines.  Routine testing using immunofluorescence with DNA stain, ELISA, or PCR is recommended to monitor possible infections.  Cells can be contaminated with bacteria (small, dark, rod-shaped that can form strings), yeast (elongated, branched structures, light with darker edges), or viruses (visible only with electron microscopy). Practical Part  Media Preparation: 1. Preheat the reagents you’re going to use to 37 °C before use. 2. Sterilize the working area using 70% alcohol. 3. Supplement the media of choice; RPMI1640 or DMEM, etc.. with 5% to 10% fetal bovine serum (FBS) and 1% antibiotic. 4. Seal the prepared media with parafilm and store at 4 °C until further use. Stem Cell Biology (BIO414) Lab 3 Culture of Stem Cells COURSE COORDINATOR: DR. SHAZA AHMED COURSE ASSISTANT: L.A. AYA EL-SHARKAWY Fall 2024 Obtaining Stem cells for Culture  There are two methods for obtaining cells: from a cell bank or by isolating cells from donor tissue. When starting culture from cells obtained from a cell bank, cells go through thawing, cell seeding and cell observation.  When using tissue collected from a donor, unnecessary tissue are usually removed if it is attached.  There are two major methods to isolate cells from the tissue: 1. Enzymatic method. 2. Explant method 1. Enzymatic method  In enzymatic method: isolation of cells from the tissue of interest using a proteolytic enzyme solution. If an enzyme is used, dilute the enzyme or stop the enzyme reaction with an enzyme reaction inhibitor, then proceed with the steps of cell seeding and cell observation to prepare the cell culture. 2. Explant method  In explant method: no enzyme is used; original tissue is excised into smaller pieces which are placed in culture dishes or flasks, and cells then start to migrate out of tissue and adhere to the culture surface Adherent vs Suspended Cells  There are two basic systems for growing cells in culture: 1. Monolayers on an artificial substrate; adherent culture; cells are anchorage- dependent and must be cultured while attached to a solid or semi-solid substrate 2. Free-floating in the culture medium; suspension culture. Manipulation of Stem Cells 1. Cell passaging (Splitting)  Passaging(also known as sub-culture or splitting cells) involves transferring a small number of cells into a new vessel.  Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated with prolonged high cell density. 2. Media change (Feeding)  In the case of adherent cultures, the media can be removed directly by aspiration, and then is replaced.  Media changes in non-adherent cultures involve centrifuging the culture and resuspending the cells in fresh media. 3. Differentiation and Transduction  Involves the process of differentiation of a cell where a less specialized cell undergoes development to acquire a more specialized form and function Cell Passaging/Splitting  Normal cells stop growing when they reach confluence (contact inhibition), and it takes them longer to recover when reseeded.  When cells reach ~80% confluency; 80% of the dish/flask is full, cells should be passaged/sub- cultured in order to dilute the cells allow their regrowth and division methods depends on the system of culture  In suspension Cultures cells passaged with a small amount of culture containing a few cells diluted in a larger volume of fresh media  In Monolayer Cultures cells first need to be detached; this is commonly done with a mixture of trypsin-EDTA; a small number of detached cells can then be used to seed a new culture. Some cell cultures, such as RAW cells are mechanically scraped from the surface of their vessel with rubber scrapers.  Why EDTA in Trypsin?  EDTA enhances the activity of trypsin.  Trypsin/EDTA is a combined method for detaching cells: o Trypsin cuts the adhesion proteins in cell-cell and cell-matrix interactions o EDTA is a calcium chelator, which integrins needs to interact with other proteins for cell adhesion. o no calcium = no cell adhesion. o EDTA: can decrease the clumping of cells. I. Suspension Culture System I. Feeding II. Sub-Culture 1. Collect media 1. Cells are resuspended by pipetting up and down (mixing). 2. Centrifuge for 5 min at 800 rpm 2. A portion of cells is thrown 3. Discard supernatant away/used for experiment (e.g. 4. Add 5 ml PBS and centrifuge 8ml out of 10ml) again 3. Fresh medium is added (8ml to 5. Discard the supernatant the remaining 2ml) 6. Resuspend the pellet in fresh media 7. Transfer to new flask Suspension Cell culture confluency II. Monolayer Culture System I. Sub-culture II. Feeding 1. Throw the old media 1. Throw old media 2. Wash with PBS 2. Wash with PBS 3. Add 3 ml/T75 flask Trypsin or (Trypsin- 3. Add fresh media; 10 ml in EDTA/Dispase/Collagenase) T75, 5ml in T25 4. Incubate for 5 min at Co2 incubator 5. Check cells under microscope 6. Stop the action of typsin with complete media 7. Collect culture (contains trypsin, cells and media) in a falcon tube and centrifuge for 5min at 800 rpm 8. Throw away the old media and resuspend cells in fresh medium (10ml) and add the cells to your new culture vessel Monolayer Cell Culture Confluency Practical Part 1. Preheat the previously prepared DMEM media; supplemented with 10% FBS and 1% P/S. 2. Assess the cells under the microscope. 3. According to the state of the confluency, cells should be fed or passaged. 4. In case of confluent T75 flask; cells should be passaged at a ratio 1:3 in T25 flasks. In case of confluent T25 flask; cells should be passaged at a ratio 1:2 in T25 flasks. 5. Label the flask with the number of passage, ex.: P1/P2…etc, Write the date, your initials and the name of the cell line.  Stem Cell Biology (BIO414) Lab 4 Cord Blood Banking & Cryopreservation COURSE COORDINATOR: DR. SHAZA AHMED COURSE ASSISTANT: L.A. AYA EL-SHARKAWY Fall 2024 Cord Blood Banking  Cord blood banking is when the baby's umbilical cord blood is collected and stored after delivery. Cord blood is what's left inside the baby's umbilical cord after it's cut. The baby's umbilical cord is clamped and cut shortly after birth.  Umbilical cord blood is rich in stem cells, which are valuable as they help treat many life-threatening diseases.  Cord blood banks exist to collect and store these stem cells. Healthcare providers use cord blood stem cells for transplants in sick people or for medical research. What are the uses of Cord Blood Banking?  Cord blood contains potentially lifesaving stem cells. People who need stem cell transplants benefit from babies’ cord blood. Once stem cells are transplanted into those individuals, they help make new, healthy cells.  Stem cell transplants help people with: Cancers; like leukemia and lymphoma. Bone marrow diseases requiring a transplant. Anemia; like sickle cell disease. Certain immune system disorders.  Researchers are studying cord blood to see how it can help treat other life-threatening conditions like Parkinson's disease and diabetes. Types of Umbilical Cord Blood Banking Cryopreservation  Cryopreservation is a method through which cells are frozen and stored at very low temperatures e.g. -200, maintaining their viability, until they are defrosted months or years later.  Cells are cryopreserved to minimize genetic change and avoid loss through contamination. It is best to cryopreserve cells when they are at their optimal rate of growth  For cryopreservation, a special freezing medium is used.  Freezing medium is a normal serum-supplemented medium plus 10% cryoprotectant such as DMSO or glycerol. The cryoprotectant prevents the formation of ice crystals and therefore protects the cells from rupture. Cryopreservation Adherent Cells Cell Suspension Cryovials are usually labeled with the date, cell number, passage number and cell type (and genetic modifications, if any). Cell culture media is removed, the The desired volume is directly culture is washed with PBS and trypsin is transferred into a 15 mL Falcon added to cover the cells and incubate tube. for approximately 2 min in a 37°C incubator. Culture is then resuspend in cell culture media and transferred into a 15 mL Falcon tube Cells are then centrifuged for 5 min at 850 rpm, then the supernatant is discarded. Cells are then resuspended and counted using a hemocytometer to determine their viability. Cell viability should be at least 75% for cryopreservation. Freezing media is then prepared and added to the required cell density. Important notes for cryopreservation DMSO (dimethyl sulphoxide) is not suitable for all cell types, therefore glycerol can be used as an alternative. DMSO in 10% concentration is most commonly used because it is toxic to the cells in higher concentrations. For mammalian cells, it is usually 1,000,000 cells/mL of freezing media. Cells should not be at room temperature in freezing media for more than 10 min. Cryovials are first placed into a CoolCell (at room temperature) and then put into a -80°C freezer for 24h. The CoolCell will ensure that the temperature decreases steadily by 1°C/minute. Cell Counting  Cells are counted using a hemocytometer (could be stained or not) in order to determine the cell count and viability.  To determine cell viability, trypan blue is used. It is a blue dye permeable to dead cells and impermeable to living cells. Cells stained with trypan blue imaged under the microscope Importance of Cell Counting To know the number of input cells is important for standardizing experiments and for measuring assay’s impact. To maintain cell cultures; splitting or plating cells. To prepare cells for transfection experiments. To prepare cells for downstream experiments that require accurate and consistent numbers of input cells, including qPCR, cell proliferation or viability studies. Cell Counting  The hemocytometer and the coverslip are gently cleaned with ethanol.  The falcon tube is swirled to ensure the even distribution of cells.  100 µL of cells are then taken to a new Eppendorf tube then 100 µL 0.4% Trypan Blue are added and mixed gently.  10 µL of Trypan Blue-treated cell suspension are the taken and applied to the hemocytometer.  The live, unstained cells are counted using the 10x objective lens in one set of 16 squares. Equations ◾ To calculate the cell density (Cells/mL): The average cell count from each of the sets of 16 corner squares X (104 )X dilution factor ◾ To calculate the cell viability (…..%): (Average number of viable cells /Total number of cells ) X 100 Counting Cells using Hemocytometer  https://youtu.be/pP0xERLUhyc?si=wzoBEZo2OD22S5BD Practical Part 1. After subculturing of confluent cells, pipette 100 µL of cell suspension in an Eppendorf. 2. Dilute the cell suspension by adding 100 µL of trypan blue and mix gently. 3. Load both chambers of the hemocytometer by pipetting 10 µL of the diluted cell suspension under the cover slip. 4. Place the hemocytometer under the microscope at 20x magnification. 5. Count both viable and dead cells in the 4 large quadrants. 6. Calculate the percentage of cell viability and cell density.  Stem Cell Biology (BIO414) Lab 5 Extraction of Mesenchymal Stem Cells COURSE COORDINATOR: DR. SHAZA AHMED COURSE ASSISTANT: L.A. AYA EL-SHARKAWY Fall 2024 Fates of Stem cells Differentiation of Stem Cells Mesenchymal Stem Cells (MSCs) Mesenchymal stem cells (MSCs) are fibroblastoid multipotent adult stem cells with a high capacity for self-renewal and differentiation. These cells have been isolated from several human tissues, including bone marrow, adipose tissue, umbilical cord matrix. Recently it has been shown that MSC originate from the perivascular niche, a tight network present throughout the vasculature of the whole body. Differentiation Potential of MSCs  Mesenchymal stem cells (MSCs) can differentiate into a variety of cell types, such as 1. Osteoblasts (bone cells) 2. Chondrocytes (cartilage cells) 3. Adipocytes (fat cells) 4. Other cell types, such as myocytes (muscle cells), neurocytes (nerve cells), and stromal cells.  On the other hand, hematopoietic stem cells (HSCs) primarily differentiate into blood cells and cells of the immune system, such as red blood cells, white blood cells, and platelets. Source of MSCs  MSCs can be isolated from various sources such as bone marrow, adipose tissue, umbilical cord, peripheral blood, placental tissue, synovial fluid, and dental pulp.  On the other hand, HSCs are primarily obtained from bone marrow but can also be found in small numbers in the peripheral blood. Therapeutic applications  MSCs have been studied for many therapeutic applications, including tissue repair, regenerative medicine, and cell-based therapies for various diseases.  HSCs, on the other hand, have been primarily used for treating blood disorders such as leukemia, lymphoma, and sickle cell anemia, as well as for bone marrow transplantation. Practical Part Isolation of bone marrow from mouse  (A) The mouse was placed in a 100-mm culture dish after anesthesia using paraformaldehyde, and washed with 70% ethanol for 2 minutes.  (B) Tibias and femurs were dissected; muscles, ligaments, and tendons were removed and the bones transferred onto sterile gauzes.  (C) Bones were transferred to a 100-mm sterile culture dish with 10 mL complete DMEM media.  (D) The dish was transferred into the biosafety cabinet and washed twice to flush away impurities; the two ends just below the end of the marrow cavity were excised with microdissecting scissors.  (E) An insulin needle was inserted into the bone cavity and used to slowly flush the marrow out using PBS or DMEM media. The bone cavities were washed twice again until the bones became pale.  (F) All the bone pieces were removed from the dish and the fat mass was left in the medium. Then the dish was incubated at 37°C in a 5% CO2 incubator. Differentiation media of MSCs  Differentiation medium promotes cells to change from one type to another, often a less specialized type becoming more specialized in form and function.  Examples of differentiation media: 1. Chondorgenic differentiation media: DMEM media supplemented with other components to differentiate mesenchymal stem cells into chondrocytes and cartilage tissue. 2. Osteogenic differentiation media: media that is designed to efficiently induce adipose-derived stem cells differentiation into osteoblasts (bone cells). 3. Adipogenic differentiation media: supplemented media that helps in transforming mesenchymal stem cells into adipocytes (fat tissue). Expected Result A) Mouse bone marrow cells in low- density culture B) Purified mouse mesenchymal stem cells C) Cartilage explant culture D) Confluent chondrocyte in primary culture.  Stem Cell Biology (BIO414) Lab 6 Flow Cytometry for Stem Cell Markers COURSE COORDINATOR: DR. SHAZA AHMED Fall 2024 Flow Cytometry ▪Flow cytometer a cytometric technique in which cells suspended in a fluid flow one at a time through a focus of exciting light, which is scattered in patterns characteristic to the cells and their components. ▪At the cellular scale, Flow cytometer could measure: Extrinsic: Cell size, cellular granules and photosynthetic pigments Intrinsic: Structural (DNA and RNA content) and Functional (Surface and intracellular receptors, DNA degradation and Cytoplasmic Ca+2) Flow Cytometry ▪ It is predominantly used to measure fluorescence intensity produced by fluorescent-labeled antibodies detecting proteins, or ligands that bind to specific cell-associated molecules such as propidium iodide binding to DNA. ▪ The staining procedure involves making a single-cell suspension from cell culture or tissue samples. The cells are then incubated in tubes or microtiter plates with fluorochrome-labeled antibodies and analyzed on the flow cytometer. ▪ The basic principle of flow cytometry is the passage of cells in single file in front of a laser so they can be detected, counted and sorted. ▪ When a cell suspension is run through the cytometer, sheath fluid is used to focus the cell suspension and pass the cells past the laser light one cell at a time. ▪ Light scattered from the cells or particles is detected as they go through the laser beam. Applications of flow cytometer Necrosis Vs Apoptosis Necrosis Apoptosis Cells exhibit: Exhibit nuclear condensation, Loss of membrane integrity, DNA fragmentation , cytoplasmic Swelling, knockdown of metabolic shrinkage. process A knockdown of metabolic Releasing of cellular components to processes, the surrounding environment loss of membrane integrity ,. Cell lysis into apoptotic bodies. Annexin & Apoptosis Phosphatidylserine (PS) is an integral part of the cell membrane and a phospholipid. In normal cells, phosphatidylserine (PS) PS is predominantly found across the cytosolic side of the plasma membrane. Early Apoptosis : PS moves ( flipped out) to the extracellular membrane, which then can bind with fluorescently labeled Annexin V. Viability dyes such as propidium iodide (PI) can not penetrate the plasma membrane. AnnexinAnnexin & Apoptosis & Apoptosis Late-stage apoptosis:  The plasma membrane will lose its integrity and Annexin V can then bind to cytosolic PS. Also PI can penetrate the cell.  The cell stains for both PS and PI.  Flow cytometry can then be used to identify apoptotic stages  Annexin V The data generated by flow cytometry are plotted in two-dimensional dot plots in which PI is represented versus Annexin V-FITC. These plots can be divided in four regions corresponding to: 1) viable cells which are negative to both probes (PI/FITC -/-; Q3); 2) apoptotic cells which are PI negative and Annexin positive (PI/FITC -/+; Q1); 3) late apoptotic cells which are PI and Annexin positive (PI/FITC +/+; Q2); 4) necrotic cells which are PI positive and Annexin negative (PI/FITC +/-; Q4). hESC Markers  Immunophenotyping Immunophenotyping is a powerful tool that uses flow cytometry to gain insight into the composition and dynamics of cell populations in an immune response. The technique involves identifying cell types in heterogeneous cell populations by using different fluorescently-tagged antibodies that target various CD markers. Different combinations of antibodies can be used to identify different groups and sub-groups of cells. For example, CD3 can be used as a general marker for T cells followed by other CD markers to identify regulatory T cells (CD4 and CD25), T helper cells (CD4), and cytotoxic T cells (CD8). Immunophenotyping can be used for discovery and can also be applied in clinical settings, for example to help diagnose and classify a leukemia or lymphoma, or refine the analysis of cancer cell lines by determining the presence or absence of cancer cell markers that correlate with different degrees of severity. Human peripheral blood leukocytes, stained with CD3- FITC (Clone UCHT-1) and CD5-PE (Clone UCHT-2). Double positive cells in the upper right quadrant represent 27.6% of the cell population, while 67.6% were negative for both CD5 and CD3 (lower left