Paraformaldehyde Fixation of Cells

Background

This fixation method is good for cells labeled by fluorochrome-conjugated antibodies to membrane antigens. It will stabilize the light scatter and labeling for up 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 Celsius in a fume hood, or in a 56 degree Celsius 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 Celsius. 

2.  0.5% Paraformaldehyde Working Solution. Add 10 ml. of the 2% Stock Solution to 30 ml. PBS+azide. Store at 4 degrees Celsius. 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 labeling 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. Pipette 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. 

PREPARATION OF 2% FORMALDEHYDE STOCK SOLUTION  (2 METHODS)

METHOD 1:

Formaldehyde preservative – 2% formaldehyde solution in protein-free phosphate-buffered saline (PBS).

Prepare as follows:

Add 2 g paraformaldehyde powder (e.g., Sigma, St. Louis, MO) to 100 ml of 1 X PBS. Heat to 70°C (do not exceed this temperature) in a fume hood until the paraformaldehyde goes into solution (note that this happens quickly as soon as the suspension reaches 70°C). Allow the solution to cool to room temperature. Adjust to pH 7.4 using 0.1 M NaOH or 0.1 M HCl, if needed. Filter and store at 4°C protected from light.

METHOD 2:

Formaldehyde preservative – 2% formaldehyde solution in protein-free PBS.

Prepare as follows:

10% formaldehyde* 20 ml

10 x PBS 10 ml

Distilled water 70 ml

* 10% formaldehyde solution (e.g., Polysciences, Warrington, PA, ultrapure, Cat.#04018), depolymerized paraformaldehyde, EM grade, methanol-free solution. 

Ethanol-fixation of Samples for Long-term Storage and Subsequent DNA Staining 

I. Materials 

70% Ethanol at – 20oC 

DNA Staining Buffer: 

Sodium citrate 0.25g

Triton–x 100 0.75ml

Propidium iodide 0.025g

Ribonuclease A 0.005g

Distilled water 250 ml 

II. Procedure 

1.      Place 1x106 cells from each sample into a polypropylene tube and centrifuge at 250 x g for 5 min.  

2.      Remove the supernatant as completely as possible without disturbing the pellet and add  1 ml of –20oC 70% EtOH dropwise to the cell pellet while vortexing. 

3.      Keep cells at -20oC until the day of DNA staining (cells can be stored for several weeks at -20oC). 

4.      On the day of DNA staining, take samples out of the freezer and spin them down by centrifugation at 250 x g for 5 min.  Remove the supernatant as completely as possible without disturbing the cell pellet. 

5.      Add 1 ml of DNA staining buffer to the cell pellet and vortex gently and briefly.  Keep cells for 15 min in the staining solution before acquisition on the flow cytometer. 

Commercial sources: 

Sodium citrate Cat# C7254 Sigma, St. Louis, MO

Triton-x 100 Cat# x-100         "

Ribonuclease A Cat# R4875         "

Propidium iodide Cat# 537059 Calbiochem, San Diego, CA 

For how long and on what temperature are you fixing your cells?

Dear Md.Mehadi Hasan Sohag

A fresh, 4% paraformaldehyde solution to be prepared in heated PBS. The cover slip, slide, or tissue-culture dish on which the cells were grown was rinsed with once with PBS and drained well, but not allowed to dry. The cells were fixed by incubating them in 4% paraformaldehyde in PBS for exactly 10 minutes at room temperature and the cells were washed twice with PBS. Normally, Paraformaldehyde fixation will not be stable. Excessively long incubations in PBS buffer will reverse the cross-linking and long fixing should be avoided. The fixed cells can be permeabilized by incubating in 0.2% Triton X-100 or NP-40 in PBS for 2 minutes at room temperature. Some preparations may need as long as 15 minutes also.

what microscope are you using? is your microscope good for plastic plates? 10 mins fixation in 4% formaldehyde is more than enough

 Thank you all for your kind answer.... :D

If you do not like to stain your cells after fixation, scew the condenser of your microscope downwards or use a phasecontrast lense to see the cells in bright field.

Good luck!

PFA is very insoluble but as soon as you adjust the pH and heat it up, it dissolves quite well. I dissolve 4% PFA in MILIq water instead of PBS and it works just fine. It is supposed to be fresh but I have made aliquots and frozen them, so I can thaw only the quantity I need and it also works well.

You could try fixing them in Eppendorfs and spinning them to resuspend PBS/glycine. Sometimes PFA can be a bit sticky and you have to spin a bit harder.