0.25% Trypsin-EDTA Solution

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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.
• DispaseDispase, also sold as Dispase II or neutral protease, is a zinc-dependent metalloprotease from Paenibacillus (formerly Bacillus) polymyxa, EC 3.4.24.4, used for gentle tissue dissociation and for detaching cells and epithelial sheets intact. It cleaves fibronectin and type IV collagen, degrades type I collagen only minimally, and does not cleave laminin or type V collagen, which is why it can separate an epidermis from a dermis or lift a confluent epithelial sheet off plastic without destroying cell-cell junctions. Because it is a metalloprotease requiring zinc for catalysis and calcium for stability, it is inhibited by EDTA, EGTA and 1,10-phenanthroline, and it must be diluted in a calcium-containing buffer rather than a chelator-based one.