I collect whole blood from pigs (EDTA coated tubes to avoid coagulation) and need to isolate the peripheral blood lymphocytes.
I dilute the blood 1:1 in PBS, then layer the diluted blood over Histopaque-1077 (2:1 diluted blood:Histopaque). I make sure to layer it slowly, and hold the tube with Histopaque at an angle to avoid mixing. However, very often there are red 'flakes' that start sinking to the bottom of the tube quickly after layering (see picture). The amount of flakes varies, but it occurs to some degree with most samples. Is there anything I can do to prevent this? Thanks in advance for any suggestions.
Maybe I didn't understand the question well, but it is supposed that RBC should go down, and it is what is happening. And you need to centrifuge it in order to separate lymphocyte.
Also the way you add the blood is important, please avoid to drop on the histopague and try to touch the falcon wall and have a constant flow. Also, perhaps the proportion between diluted blood and histopague could have some effects
Thanks for the feedback Mohammad Kamely !
I'm completely new at this and was worried it was a sign of poor layering. I'll have a look at the blood:Histopaque proportions as well.
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