Our lab uses a histopaque density gradient to isolate the PBMCs from whole blood. Once the cells have been isolated and are ready to be washed, I have noticed that our cells adhere very strongly to the bottom of the tube. I cannot flick the bottom of the tube to remove the PBMCs. Typically, I have to use a disposable pipette to gently resuspend the cells; however, I am worried that I am lysing the cells thus lowering their viability.
Does anyone have any suggestions as to why this may be occuring?
Also, we typically assess viability using a 1:1 trypan blue stain with the Countess II. We have noticed that our viability is not very high and is often variable. Other labs in our department use the PBMC isolation tubes and achieve similar viability when using the same protocol (i.e., 1:1 trypan), so this leads me to believe that it is not our protocol. The viability is also >90% when counting with a hemocytometer.
Has anyone had success counting using the Countess II and a trypan blue stain?
Hi. Theodore. Have you tried using lower centrifugation speed or time? It did help me but did not resolve the issue completely. Hope it will help.
I agree with Deven Patel . We isolate PBMC and spin at lower speeds 300 - 400g for the washes. This also minimizes platelet contamination and doesn't affect viability much. Instead of tapping, gently bring the pellet into the solution with the help of 1mL pipettors/disposable pipettes.
Pragun Muthya & Deven Patel
Thanks for the responses! We have currently been washing at 500g. I can try to lower the speed and see if that helps. We have been using disposable pipettes to re-suspend the pellet because we were not successfully re-suspending it by flicking it.
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