Trypan blue will stain dead or dying cells. Viable cells are able to repell the dye and do not stain. Note: Trypan blue has a greater affinity for serum proteins than for cellular protein. If the background is too dark, cells should be pelleted and resuspended in protein-free medium or salt solution prior to counting. Supplies & Equipment: Eppendorf tubes (1.5 ml) Micropipet (10µl) Hemocytometer Reagents: HBSS (Hanks' Balanced Salt Solution) sterile Trypan blue solution 0.4% (Sigma T-8154) Procedure: Prepare a cell suspension in HBSS Transfer into Eppendorf tube: 0.5 ml of 0.4% Trypan blue solution 0.3 ml of HBSS 0.2 ml of cell suspension in HBSS (= dilution 1 : 5) Allow to stand for 5 to 15 minutes Note: after prolonged incubation, viable cells start to take up dye as well. Pipet 10µl of this mix into cover-slipped chambers of hemocytometer Note: avoid cell clusters by pipetting up and down. Count viable and non-viable cells Note: for optimal results, adjust cell density to 20-50 cells / square. Calculations: cells/ml: the number of cells per quadrant equals 104 cells / ml (e.g. 50 cells per quadrant = 0.50 million cells / ml) total cells: cells / ml x original volume (e.g. 5 million cells in 10 ml) cell viability (%): total viable cells (unstained) / total cells (stained and unstained) x 100 (e.g. 25 stained cells per quadrant: 50% viability) |
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