Hello Arturo Mamani
When you obtain a PBMC pellet that is red in color, it means that PBMCs have been contaminated with RBCs. Care should be taken not to disturb the erythrocyte layer upon removal of PBMCs. A red PBMC pellet means that RBCs must be lysed prior to further PBMC processing lest you risk their presence altering the results of your assay.
For RBC lysis, it is necessary to resuspend the PBMC pellet in 1 mL of Red Cell Lysis Buffer and place in the dark at room temperature for 20 min. After lysis is complete, centrifuge the cells at 500 rcf for 5 min, remove the supernatant, resuspend the pellet in 1 mL PBS, and repeat the centrifugation to wash the cells prior to proceeding to the next step.
Recipe for Red Blood Cell Lysis Solution.
0.15 M ammonium chloride
10 mM potassium bicarbonate
0.1 mM EDTA
Prepare with distilled H2O. Filter through a 0.45µm filter. Store at room temperature.
Best.
As Malcolm Nobre said, red pellet means RBC contamination. I've been working with PBMC a lot and I can say it happens even when we are carefully removing the PBMC layer. Sometimes, it simply gets contaminated. On the other hand, RBCs in your PBMC are not necessarily a problem. We are cryoconserving them, culturing them, stimulating them etc and that initial RBC contamination was not a problem so far. RBCs get lost in the process, especially if you cryoconserve them or go through multiple culture passages. However, it all comes down to your downstream application and that's what you have to consider.
Totally agree with the comments on RBC contamination. I will note that when a patient is anemic and or myelosuppressed they can have an increased frequency of reticulocytes that don't lysis well. I will also note that under these physiologic conditions, myelopoiesis can result in circulating myeloid precursors, i.e. a left shift, and a moderate to high frequency of hypodense myeloid cells contaminating the mononuclear cell inter-phase layer following F/H separation.
I can also add that it is not at all uncommon for post-Ficoll PBMC from umbilical cord blood to contain reticulocytes, causing the pellet to be pink to red. If you store the cells frozen, the reticulocytes will not survive the freezing process, so they will be gone when you thaw the cells. Alternately if you are purifying specific cell populations from the PBMC, the reticulocytes are frequently lost during the selection process.
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