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Protocol: Trypan Blue Method
Sampling
| Cell Density ( 10^6 ml) | Dilution | PBS ( µl ) | Cells ( µl ) | Trypan Blue ( µl) |
| 1-2 | 4 | 100 | 50 | 50 |
| 2-4.5 | 8 | 125 | 25 | 50 |
| 4.5-7 | 12 | 120 | 15 | 45 |
| >7 | 16 | 137.5 | 12.5 | 50 |
- Take xµL ofPBS in 96 well plate
- Add required volume of cell culture. Mix once
- Bring the plate back to microscope, add trypan blue to the PBS + Cell mixture just before counting the sample.
Trypan Blue is toxic to cells. If left too long with trypan blue, even healthy cells will die.
Calculation of LCD :
LCD = Cell Count/ ( 100* 4) * Dilution
Tips :
- Mix cells before sampling
- Take cell sample from top of the liquid
- Mix trepan blue into the PBS + cel solution slowly and well before loading
