ICBR Flow Cytometry Core Laboratory
Paraformaldehyde Fixation of Cells
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.
- 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.
- 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.
- 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.
- pH meter. This is involved in making the paraformaldehyde stock solution.
- Balance with a resolution of at least 0.1 g. Again, this is to make the paraformaldehyde stock solution.
- One liter graduated cylinder or volumetric flask. Ditto.
- Centrifuge. You should know how the RPM translates into G-force.
- Pipet in the range of 500 to 1000 microliters (0.5-1.0 ml.).
- Vortex mixer. You could mix by tapping or shaking the tubes, but a mixer will give much more reproducible results in most cases.
- Refrigerator. To store the preserved cells.
Analyze the cells on the flow cytometer within one week.
- Following the last wash step, centrifuge the cells and remove the liquid, as described in the direct or indirect antibody labelling procedure.
- Add 0.5 to 1.0 ml. of cold 0.5% paraformaldehyde solution. Vortex immediately.
- Store the cell suspension at 4 degrees celcius in the dark.
- Lanier, L.L., and Warner, N.L.: Paraformaldehyde fixation of hematopoietic cells for quantitative flow cytometry (FACS) analysis. J. Immunol. Meth., 47:25, 1981.
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