0.05% Trypsin-EDTA Solution, 500 mL

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• Counting Cells with a HemocytometerA hemocytometer is a thick glass slide bearing a precisely etched grid over which a coverslip creates a chamber exactly 0.1 mm deep, so that a known volume of cell suspension can be counted under a microscope and converted to a concentration. For mammalian cells you count the four 1 mm2 corner squares, take the mean, multiply by the dilution factor, and multiply by 10^4 to obtain cells per mL, because each corner square encloses exactly 10^-4 mL. Mixing the sample 1:1 with 0.4% trypan blue before loading gives a viability percentage from the same count, since only dead cells with compromised membranes take up the dye.
• Trypsin-EDTATrypsin-EDTA is a cell dissociation reagent that combines the serine protease trypsin, which cleaves peptide bonds on the C-terminal side of lysine and arginine residues in cell-surface and matrix proteins, with the chelator EDTA, which binds the calcium and magnesium ions that cell-adhesion molecules require. It is supplied in a calcium- and magnesium-free balanced salt solution, most commonly at 0.05% trypsin (0.5 g/L) for routine cell lines and 0.25% (2.5 g/L) for firmly adherent cells and primary cultures. Typical use is 2-5 minutes at 37 degrees C, followed immediately by neutralisation with serum-containing medium or a defined trypsin inhibitor.
• Subculture of Cells (Passaging)Subculture of cells, also called passaging, is the transfer of cells from a culture that is approaching confluence into fresh vessels with fresh medium so that growth can continue. Adherent cells are detached first, usually with 0.25% or 0.05% trypsin-EDTA or a non-enzymatic dissociation reagent, then reseeded at a lower density set by a split ratio such as 1:4; suspension cells are simply diluted into fresh medium without any dissociation step. Most continuous adherent cell lines are subcultured at 70-80% confluence, which normally means two or three passages per week.
• Trypsin vs Accutase: Choosing a Cell Dissociation ReagentTrypsin is a pancreatic serine protease that cleaves peptide bonds after lysine and arginine, and it detaches cells fast but indiscriminately - it must be stopped with serum or a trypsin inhibitor, and it cleaves surface proteins along with the adhesion contacts. Accutase is a proprietary mixture of proteolytic and collagenolytic enzymes from an invertebrate source, supplied in calcium- and magnesium-free DPBS with 0.5 mM EDTA; it works more slowly and gently, does not require a neutralisation step, and preserves more surface epitopes. Choose trypsin for routine passaging of robust adherent lines where speed matters and the cells go straight back into culture; choose Accutase when the harvested cells are the experiment - flow cytometry on surface markers, pluripotent stem cells, primary neurons, or any single-cell suspension where viability and phenotype must survive the harvest.
• Poly-L-Lysine Coating for Cell CulturePoly-L-lysine coating covers a culture surface with a positively charged synthetic polymer that binds the negatively charged plasma membrane, allowing weakly adherent cells to attach to glass or plastic. The standard protocol applies a sterile 0.01% (0.1 mg/mL) solution at roughly 1 mL per 25 cm2, leaves it in contact with the surface for 5 minutes to 1 hour at room temperature, aspirates, rinses thoroughly with sterile tissue-culture-grade water, and air-dries the surface for at least 2 hours before cells are seeded. The attachment it provides is purely electrostatic: poly-L-lysine does not engage integrins, so cells that need genuine extracellular matrix signalling require laminin, fibronectin or collagen instead of it or on top of it.