Ki - 67 Staining Protocol
Prepare 70 % ethanol and chill at - 20°C .
Prepare target cells of interest and wash 2X with PBS by centrifuge at 350xg for 5 minutes .
Discard supernatant and loosen the cell pellet by vortexing .
Add 3 ml cold 70 % ethanol drop by drop to the cell pellet while vortexing .
Continue vortexing for 30 seconds and then incubate at - 20°C for 1 hour .
Wash 3X with BioLegend’s Cell Staining Buffer ( Cat . No . 420201 ) and then resuspend the cells at the concentration of 0.5 - 10 x 106 / ml .
Mix 100 µl cell suspension with proper fluorochrome - conjugated Ki - 67 antibody and incubate at room temperature in the dark for 30 minutes .
Wash 2X with BioLegend’s Cell Staining Buffer ( Cat . No . 420201 ) and then resuspend in 0.5 ml cell staining buffer for flow cytometric analysis .
