Hi, I'm trying to fractionate blood components by density centrifugation. I've been able to clearly isolate the monocyte/lymphocyte layer, however the granulocytes tend to drift off into the packed RBC layer. The protocol includes using 100 ul of blood in a glass capillary tube and centrifuging the sample at 1500 RCF for 5 min.
Any suggestions on how to prevent this from happening without the addition of density gradient medium? Is it because the the centrifugation force is too high or the centrifugation time is too long?
Thanks for your help!
Well, I'm afraid if this was easy to do without gradient... Nobody would be using gradients :) My group tried that kind of protocol and couldn't do it reliably either. We settled on the polymorphprep reagent, which can be easily removed from your granulocytes by repeated centrifugations once they are pure. It's also visible on Giemsa smears, so you can tell if it's still there in your final preps.
Also, the chance of this is low, but it can happen: I had a donor once with asymptomatic heterozygous Thalassemia. Her erythrocytes were lighter than normal and co-mingled with the granulocytes even on a density gradient. So try switching your donor, and a miracle might still happen.
Decrease the speed of centrifugador. I have seen monolayer clear in sedimentation rate tube. Wintroble model.
The speed seems too high . i think reducing the speed will help. use a collect centrifuge that is designed to separate blood components. do this at different levels and you will finally get there.
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