During PBMC separation, my samples did not form any PBMC ring in the interphase. Rather the PBMC formed a lining just on top and along with RBC. It is clearly visible in the image.
Please note that I have used Alsever's solution as an anticoagulant while collecting blood and PBMC separation procedure was done after 5 days of collection. In the meantime, the samples were in a 2-8 degree refrigerator.
Could it be the tubes were not balanced correctly in Centrifuge? or you did not decelerate slowly enough
Hello K M Main Uddin
Additionally, I would like to mention.
How have you loaded the diluted blood sample on the density gradient solution?
It is important that the diluted blood should not mix with the density gradient solution. The blood should layer over the density gradient solution and slowly rise until all the blood is on the top of the density gradient solution. Minimize the amount of mixing at the interface for optimal separation of blood by carefully layering the blood, by placing the pipette tip close to the density gradient solution and allow the blood to run down the side of the tube slowly. Centrifuge the layered mix at 400g for 30 mins without brakes. After centrifugation you should get 5 layers from top to bottom as:
plasma,
buffy coat containing PBMCs,
density gradient material ( for instance, Ficoll-Paque),
granulocytes and
erythrocytes.
I have never used Alsever's solution. I would not be in a position to comment on the viability of PBMCs after 5 days of storage at 2-8 degree C. Usually, PBMCs are separated within 6-8 hrs after the withdrawal of blood.
Best.
Hello, K M Main Uddin!
You should verify the temperature during the centrifugation steps. I never tried to isolate PBMC after long periods of storage, but I think it may result in a poor cell recovery.
You can also chek this guide for troubleshooting.
All the best
PBMC should separate within 24 hours of collection if you want to recover it in cell culture. Please check the acceleration and deceleration speed, it should be zero. Also, you can try with a lower final speed (300-400 G) than the general 500G speed for Alsever's solution.
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