I’m trying to isolate lymphocytes from human blood using histopaque. First I added 10ml of histopaque in a 50ml falcon tube then I layer 10ml blood on top of it. The tube was centrifuged at 6000rpm for 45 minutes. I am not able to get buffy coat, im attaching the pictures of the media and blood in a falcon after 45 minutes of centrifugation. Kindly help.
Hello Mehar Khalil
You should try to do the following.
You need to dilute the blood with equal amount of PBS. Then mix the blood and PBS gently. When erythrocytes in whole blood are aggregated, some mononuclear cells are trapped in the clumps and therefore sediment with the erythrocytes. This tendency to trap mononuclear cells is reduced when you dilute blood with PBS.
Add 14 ml histopaque at the bottom of a 50 ml tube without touching the side of the tube. Gently overlay the Histopaque with the diluted blood (20ml). Allow the diluted blood to flow down the side of the tube and pool on top of the density gradient media surface without breaking surface plane. Centrifuge at room temperature at 760 x g for 30 min with the brakes off since the deceleration can disrupt the density gradient.
Best.
Thank you Malcolm Nobre . looking at the pictures, what do u think has gone wrong? Secondly, if the blood was collected 12 hours before isolation and stored in an EDTA tube, does that also effect the isolation of lymphocytes?
Mehar Khalil perform the isolation of lymphocytes less than 8 hours after blood collection. You can get good results if the procedure is performed less than 2 hours after blood collection. Lymphocyte isolation even 12 hrs. after blood collection should not be a problem. As mentioned above I feel you should perform centrifugation at 760 x g for 30 min instead of 6000rpm.
In our laboratory, we first mixed the whole blood with 1:1 PBS or Serum-free media. We slowly layer it over the histopaque (usually 600 ul for ~5 ml of blood) and centrifuged it at 2500 rpm for 20 minutes. Following, the top buffy layer is aspirated and mixed with 3-5 ml of PBS/ Media and centrifuged at 2500 rpm for 10 minutes to pellet the lymphocytes.
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