I am interested in isolating naïve CD4+ T cells from human PBMCs. I get whole blood (~500 mL blood + 70 mL anticoagulant) from a blood center but I don't receive it till +1 day after extraction. Blood sample is stored and delivered on ice. Once I get the sample, I start performing the Ficoll gradient. However, the amount of PBMCs that I have obtained (~0.25*10^6 cells/mL) is lower than expected (~10^6 cells/mL - this is the amount I used to obtain using fresh whole blood samples). I have been reading about keeping blood samples at room temperature before processing, does anyone have experience with this approach?
On the other hand, does anyone know about the percentage of naïve CD4+ T cells in the PBMCs fraction? Thanks!
Hello Laura Calvo-Barreiro
I have been reading about keeping blood samples at room temperature before processing, does anyone have experience with this approach?
From healthy blood, PBMC yield ranges between 0.5 - 3 x 10^6 cells per mL blood. If storing is needed before processing, store the blood at room temperature in the dark and on a rocker. Always keep the blood samples at 20-37 degree C, do not refrigerate the blood samples. Refrigeration of blood does not generally introduce variation in full blood counts but has been reported to modify representation of multiple lymphocyte subsets within PBMCs. When blood samples are held at room temperature, generally considered to be in the range of 20–37 degree C, time-to-processing is another factor that may impact PBMC quality. So, process the blood sample immediately when it arrives.
Does anyone know about the percentage of naïve CD4+ T cells in the PBMCs fraction?
The percentage of naïve CD+ T cells will vary between individuals depending on the age and the immunological status. T cells comprise, approximately, 45–70 % of PBMCs in human peripheral blood, with a count in healthy individual in the range of 1 million cells/mL.
Best.
Thank you Malcolm Nobre.
Do you have experience processing buffy coats instead of whole blood to obtain PBMCs? If you do, how do you dilute the buffy coat before adding it on top of the Ficoll? How long and which g-force you use in the centrifugation steps? Thanks!
Hi
I have read your message and I was surprised that you get blood stored on ice. When we did our experiments at University of Liverpool, we used to have fresh blood.
Please find the attached file for more info.
Best regards
Hi,
I have tried both and I clearly prefer room temperature. Ficoll should also be heated (37°C) just before use, as well as new culture medium. 24h storage is OK to get PBMCs.
Best regards
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