I have been differentiating K562 cells and would like to count the differentiated cells using a haemocytometer. I have tried SIGMAFAST DAB as well as o-diansidine.
mix 14.6ml acetic acid with 485.4ml dH2O and solve 1g benzidine dihydrochloride (keep this in dark, e.g. covering in foil)
For staining: mix 1ml of this mixture with with the same volume of cell suspension and add 1µl H2O2 (2%). Incubate for 1-2min at rt and after incubation you can measure your probes.
mix 14.6ml acetic acid with 485.4ml dH2O and solve 1g benzidine dihydrochloride (keep this in dark, e.g. covering in foil)
For staining: mix 1ml of this mixture with with the same volume of cell suspension and add 1µl H2O2 (2%). Incubate for 1-2min at rt and after incubation you can measure your probes.
2. To prepare a 0.2% TMB, dissolve 2mg/mL TMB in 0.5% acetic acid
Pr 50mL: Dissolve 100mg in 50mL 0.5% acetic acid. This can be stored @ 4C for up to 1 month.
3. Prepare just before use: Add 5ul of 30% hydrogen peroxide/1mL of 0.2% TMB.
Pr 10mL: Add 50ul of 30% hydrogen peroxide
Staining:
Wash K562 cells once with PBS and resuspend in 0.25-0.5 mL of PBS. Add 1 volume (0.25-0.5 ml) of 0.2% tetramethylbenzidine working solution and incubate 10-30 minutes in the dark at room temperature (it’s toxic, so do not incubate longer than 30 min in total including picture taking). Count brown cells in light microscope