I am isolating PBMCs from whole blood by Density Gradient Centrifugation. have used Lymphoprep and a Leucosep tube, and after centrifuging the sample, the layers in the tube were more red than usual. Then, when I recovered the PBMCs layer and put it in another tube and wash it, the pellet was very big and red. I lysed the red blood cells with 5ml of red blood cells lysis buffer for 15 min. However, after washing and counting the PBMCs I have counted 120x106 cells, that came from a total volume of whole blood of 16 ml, so I think it is impossible to have so many PBMCs in that volume. Could it be that it is not only PBMCs but also red blood cells or granulocytes? I have already frozen the cells, and I will use them to do flow cytometry. When I thaw them, should I incubate again with red blood cells lysis buffer before staining? any other recommendation?
Thank you in advance!
Hello Cristina Crespo
If PBMCs are cryopreserved and stored in the gaseous phase of liquid nitrogen prior to use, the red blood cells do not survive these temperatures and are no longer a problem upon thawing of the sample. However, if you use fresh PBMCs for a given assay, contaminating red blood cells would result in an erroneous cell count. You may avoid this, by diluting PBMC samples in glacial acetic acid (which lyses RBCs) if you must count them manually.
So, it is possible that the count, 120 x 10^6 cells, could include PBMCs as well as red blood cells or granulocytes.
On the other hand, granulocyte contamination is more challenging. Granulocytes have the shortest half-life of all blood cells. Therefore, as the blood ages i.e., between collection time and use, they begin to change density which may reduce the effectiveness of a density gradient separation. Also, with a half-life of approximately 9 hours, the granulocytes can die and release products which are known to alter the function of certain populations including the T cells. Granulocytes too do not survive cryopreservation and storage in the gaseous phase of liquid nitrogen. But upon thawing, if PBMCs are contaminated with granulocytes, it may yield a poor cell viability.
You need not incubate PBMCs again with RBC lysis buffer upon thawing.
Best.
Malcolm Nobre Thank you very much for your reply. I am freezing the cells in liquid nitrogen for long term storage. So then I will not have to worry about RBC.
For granulocytes, there is nothing I can do about it if I already froze the samples then? I suppose I can still not gate them when I do flow cytometry?
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