I have some samples of frozen PBMCs (peripheral blood mononuclear cells) that I am running through a flow cytometer setup. The cells are frozen using a standard DMSO based protocol. When unfreezing them I put them in a 37 C water bath and then dilute with PBS 1X in the centrifuge at 400 g for 10 minutes. However, once I do this and get a pellet at the bottom of the falcon tube it seems like I can't really do anything else with them or I'll lyse most of the cellular pellet.
The reason this is an issue is that many times the cell pellet contains visible red pigment (red blood cells/hemoglobin). I would like to remove this pigmentation, however, if I run an additional ficoll protocol the cells do not centrifuge correctly (some end up at the bottom of the histopaque while others float in the middle of the solution, etc.) and if I use RBC lysis buffer 90% of my pellet lyses. (I'm guessing this is because the cells are in a poor state after unfreezing, etc.)
What can I do to prevent the cells from lysing while still being able to remove the RBCs?
Specific protocols would be nice, btw. ;)
Hi Benjamin, procedures to address red cell contamination are often addressed prior to freezing. In your thawing process you'll likely benefit from a more gentle method of cell handling. Most thawing procedures for PMBCs involve "complete medium" preparations and may often include a nuclease cocktail to prevent some clotting brought on by lysis of some of the thawing cells. If you are going to do any other preps for the cytometry aspect, hopefully the red cells will not be so overwhelming as to completely occlude your other cell types in the data collection phase. They will have little or no affect on the efficiency of standard antibody-based staining. See the following as a starting point:
Weinberg, A., Song, L. Y., Wilkening, C., Sevin, A., Blais, B., Louzao, R., … Fenton, T. (2009). Optimization and limitations of use of cryopreserved peripheral blood mononuclear cells for functional and phenotypic T-cell characterization. Clinical and Vaccine Immunology, 16(8), 1176–1186. http://doi.org/10.1128/CVI.00342-08
Hi Benjamin,
I agree with Sam, thawing the cells in PBS is to stressfull for them in my experience.
I had the best results with thawing them briefly at 37° (until a pea sized piece of ice remains), transfering them to 2 mL of medium with 50% FBS (cold) and filling the tube up to 15 mL with medium containing 15% FBS (cold). Centrifuge at 4^for 5-8 minutes at 300g. Resuspend in medium with 30% FBS and centrifuge again. After this they are ok in PBS.
The last couple of times this gave me a viability between 60-65% and they perfomed well in FACS.
I would try to remove any erythrocyte conamination before freezing, especially to keep the stress as low as possible for the cells.
- Badges
- Science topic
- Similar topics
- Engineering
- Materials Engineering