Flow Cytometry Core Fixation of Cells :: Nationwide Children's Hospital

Paraformaldehyde 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

1. 2X Paraformaldehyde Stock Solution

  • Add 2 g 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.

2. 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.

3. Antibody-labeled cells in PBS+azide at a concentration of  1 million cells/ml.

Procedure

1. After the last wash step in the staining protocol centrifuge the cells and remove the liquid.

2. Add 0.5 to 1.0 ml. of cold 0.5% paraformaldehyde solution and vortex to avoid clumping.

3. Store the cell suspension at 4 degrees Celsius in the dark and analyze within one week.

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