I have been separating PBMC from peripheral blood on lymphoprep at 1000g, 30 minutes with no break. Some patients' blood separates very clearly while some have either a cloudy lymphoprep layer with white percipitance/clumps present, or having haemolysis/red blood cells in the PBMC layer.
How do I get rid of haemolysis or presence of red blood cells in the PBMC layer and what do the white clumps represent?
Hi Yusuf,
in our experience, 1000g is too fast for gradient centrifugation of blood sample. We usually perform PBMNC isolation at 400g...
Is that with healthy controls or patient's blood? And how well of a separation do you get?
This suggested protocol runs perfectly, equal for both patients and controls. Good tests!!
Hi Yusuf,
I agree with the previous two posts regarding the speed and time. Not sure if you already do this, but do you dilute your blood sample prior to layering on the Lymphoprep? We especially need to dilute the blood if we're working with apheresis samples. We also use ACK Lysis buffer if there's remnant red blood cells in the PBMC layer after isolation. When we do use the lysis buffer, we wash the pellet with 10x vol of the lysis buffer and at least two times. Good luck!
Hi Norkhairin,
no matter the speed you are using or brake. Somehow, sometimes, ficoll gradient gives the results you mentioned. I can just say that you can predict the outcome by resting the anticoagulated blood tube and looking if, when the plasma naturally separates from decanting RBC and leukocytes, it remains somewhat red and not clearly yellow. I think it depends on donor health status and/or anticoagulation efficiency.
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