After PBMC isolation and washing, I count the cells of PBMC. Most of the time RBCs exist in the pellet and when I count the total PBMC I am not sure if the counted cells were RBCs or lymphocytes. If RBCs are also stained by Trypan Blue can we distinguish them from other cells to not count them?
Hello Ahmed Al-Omar
As far as possible avoid RBC contamination while isolating PBMCs as it may lead to inaccurate counts. This is most likely to be a problem for samples which are left overnight before processing, because these samples tend to have less separation between PBMC and RBC in the Ficoll gradient. After isolating PBMCs, you can use RBC lysis buffer. Lysis with ACK (ammonium-chloride-potassium) buffer can reduce the frequency of RBC in the sample.
If you still get RBC contamination, you may prepare a 1:1 mixture of Trypan Blue and 2% acetic acid that can be used to dilute the PBMC cell suspension. The acetic acid will lyse red blood cells that may be present in the sample.
Best.
Hello,
First of all, I think there's room to improve your PBMC isolation technique. There usually are very little RBCs left after a proper isolation of PBMC.
Yes, RBCs can be positive to Trypan blue dye. You can differentiate between RBCs and WBCs under the microscope, you just need some practice. You can try phase contrast microscopy which can help you distinguish between the two.
A simpler approach is to take a small sample of your suspension and analyze it with an automatic hematology analyzer, just like you would do with a whole blood specimen for CBC. Some analyzers will reject PBMC suspensions because they don't meet the blood criteria, but you can usually turn this off in the device's settings.
As Malcolm mentioned, you can always try to lyse the remaining RBCs in your PBMC suspension. However, RBC lysis will stress the PBMCs. Whether RBC lysis is a good idea or not pretty much depends on the desired downstream application of your PBMCs.
Good luck!
RBC do stain with trypan blue. You can avoid RBC contamination by using an RBC lysis buffer or isolating your PBMCs using gradient centrifugation. If you are using an automated cell counter there really isn't a way to avoid them being counted once they are in your sample.
Malcolm Nobre Ion Bogdan Mănescu Kevin Muñoz Forti Thank you very much for your answers.
Hello Ahmed, RBC may pick up TB staining but they are significantly smaller and without a nucleus so with a little experience differential counting should be easier. If using a lysis solution check that it does not contain fixative and that your cells are not effected biologically. I found exposure to ammonium chloride solutions for rbc suppressed NK activity. I had to use osmotic shock to clear rbc contamination if I wanted to retain cell function.
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