Ki-67 Flow Cytometry Staining Protocol

Protocol Steps

1. Prepare 70% Ethanol and chill to -20°C
   Tip: Do not freeze ethanol for long-term storage.  


2. Prepare target cells of interest and wash 2X with PBS, centrifuging at 350xg for 5 minutes.


3. Discard supernatant and loosen the cell pellet by vortexing.


4. Add 3ml cold 70% ethanol drop by drop to the cell pellet while vortexing.


5. Continue vortexing for 30 seconds and then incubate at -20°C for 1 hour.


6. 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.


7. Mix 100µl cell suspension with proper fluorochrome-conjugated Ki-67 antibody and incubate at room temperature in the dark for 30 minutes.


8. Wash 2X with BioLegend's Cell Staining Buffer and then resuspend in 0.5ml cell staining buffer for fluorescence activated cell sorting (FACS), or flow cytometric analysis.
   Tip: Based on customer testing, Ki-67 staining is not recommended with our True-Nuclear™ Transcription Factor Buffer Set.