Hi,
Some of the samples i processed have a red layer beneath the buffy coat and compromise greatly my cell count. Can it be safe to add a red blood cell lysing step to my PBMC isolation? Has anyone encounter such problems?Please advise.
Hello Berenice. It is perfectly safe to lyse the erythrocytes that stick to the PBMC layer. You can use red cell lysing buffers (containing NH4Cl, for instance) 5 min on ice and your PBMC would be fine. It is rather common to find such phenomenon, it may be associated with the platelet numbers in each sample.
Esmeralda
Red cell lysing buffers do have an effect although minimal on your PBMC so it is depending on what you would like to do with the isolated cells. How do you count the cells? because if it's just to be sure of your actual number you could change the counting soultion. For example if you normally use trypan blue for cell counting in a counting chamber you can use Türk solution instead, which lysis red blood cells present in the sample.
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