Fixation of Cells

This fixation method is good for cells labeled by fluorochrome-conjugated antibodies to membrane antigens. In addition to stabilizing the scatter properties of cells it will also inactivate most biohazardous agents.

Materials

  • 2X Paraformaldehyde Stock Solution
    • Add 2g paraformaldehyde to 100 ml PBS+azide.
    • Heat to 70o C in a fume hood just to the point that the paraformaldehyde goes into solution.
    • Allow to cool to room temperature and then adjust to pH 7.4.
    • Store at 4o C.
  • 0.5% Paraformaldehyde Working Solution
    • Add 10 ml. of the 2% Stock Solution to 30 ml PBS+azide. Store at 4o C. This solution is stable for up to 1 week.
  • Antibody-labeled cells in PBS+azide at a concentration of 1 million cells/ml.

Procedure

  • After the last wash step in the staining protocol centrifuge the cells and remove the liquid.
  • Add 0.5 to 1.0 ml. of cold 0.5% paraformaldehyde solution and vortex to avoid clumping.
  • Store the cell suspension at 4 degrees Celsius in the dark and analyze within one week.