Cell Fixation and Permeabilization Protocol using 70% Ethanol

Protocol Steps

Cell Fixation and Permeabilization Protocol Using 70% Ethanol

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 2X with BioLegend Cell Staining Buffer (Cat. 420201) and resuspend cells at 0.5-1.0 x 107 cells/ml.  


7. Use 100µl cell suspension/staining tube.
   Note: Please note that certain markers or fluors may not survive ethanol fixation. Protein-based fluors, like PE and APC, tend to have more difficulty, while synthetic fluors, like Brilliant Violet™, tend to have a higher chance of surviving the process.