ICBRFlowCytometryCoreLaboratoryParaformaldehydeFixationofCells

Background

This fixation method is good for cells labelled by fluorochrome-conjugated antibodies to membrane antigens. It will stabilize the light scatter and labelling for up to to a week in most instances, allowing you to be more flexible in scheduling cytometer time. Furthermore, it inactivates most biohazardous agents, so it is important from a safety standpoint as well.

The procedure picks up at the end of the direct or indirect staining procedures. The cells are expected to be in 12 x 75 mm. plastic culture tubes, one million cells per tube.

It should not be used with the procedure to label dead cells. Fixed cells have a permeable membrane - the dye would enter all the cells.

Materials

1. 2X Paraformaldehyde Stock Solution.

   
   

   
   

• Add 2 g. paraformaldehyde to 100 ml. PBS+azide.

• Heat to 70 degrees celcius in a fume hood, or in a 56 degree celcius water bath, just until the paraformaldehyde goes into solution.

• Allow to cool to room temperature, then adjust to pH 7.4 using 0.1 M. NaOH or 0.1 M. HCl, as needed.

• Store at 4 degrees celcius.

2. 0.5% Paraformaldehyde Working Solution. Add 10 ml. of the 2% Stock Solution to 30 ml. PBS+azide. Store at 4 degrees celcius. This solution is stable for up to 1 week.

   
   

3. Antibody-labeled cells in PBS+azide. They may have been labeled using either the direct or indirect labelling procedures. Concentration should be 1 million cells in 1 ml.

   
   

Equipment

1. pH meter. This is involved in making the paraformaldehyde stock solution.

   
   

2. Balance with a resolution of at least 0.1 g. Again, this is to make the paraformaldehyde stock solution.

   
   

3. One liter graduated cylinder or volumetric flask. Ditto.

   
   

4. Centrifuge. You should know how the RPM translates into G-force.

   
   

5. Pipet in the range of 500 to 1000 microliters (0.5-1.0 ml.).

   
   

6. Vortex mixer. You could mix by tapping or shaking the tubes, but a mixer will give much more reproducible results in most cases.

   
   

7. Refrigerator. To store the preserved cells.

Procedure

1. Following the last wash step, centrifuge the cells and remove the liquid, as described in the direct or indirect antibody labelling procedure.

   
   

2. Add 0.5 to 1.0 ml. of cold 0.5% paraformaldehyde solution. Vortex immediately.

   
   

3. Store the cell suspension at 4 degrees celcius in the dark.

   
   

Analyze the cells on the flow cytometer within one week.