0.5 mM EDTA Solution, Calcium/Magnesium-Free

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• Trypan Blue and the Dye Exclusion Viability AssayTrypan blue is a diazo dye used at 0.4% (w/v) to separate live from dead cells by dye exclusion: an intact plasma membrane keeps the charged dye out, so viable cells stay clear and refractile, while cells with a damaged membrane take the dye up and stain blue. In practice the cell suspension is mixed 1:1 with 0.4% trypan blue, loaded into a hemocytometer or an automated cell counter, and counted within 3-5 minutes to give both a cell concentration and a percent viability. Because it reports membrane integrity only, trypan blue counts cells that have already died -- it does not detect apoptotic or metabolically failing cells that still have an intact membrane, so it reads high compared with metabolic or flow-cytometry viability assays.
• 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.