Hi,
I am isolating PBMC from undiluted human blood using histopaque 1119 and 1077(Sigma). In our system, we can not dilute the blood because simultaneously we need plasma in original concentration. As a result we are unable to get the clear separation at the 1077-1119 interface. the lower layer containing granulocytes are merged with RBCs.
So how to resolve this problem?
I have got one reference that uses 2:1 histopaque vs blood ratio(example 2ml 1119-2 ml 1077-2ml of blood). Usinng this method I have got the clear separation. But this article was for Dog blood.
So my question is:
1. Can we use 2:1 ratio for histopaque vs blood?
2. Is there any reference to support this protocol for human blood.
Reference: A simple method for the simultaneous separation of peripheral blood mononuclear and polymorphonuclear cells in the dog, Veterinary Immunology and Immunopathology
62 -1998. 29–35
Hello.
Sorry for the late answer. I had similar problems with granulocytes and RBC. Did you find the way to solve your problem? In our lab we tried to increase histopaque 1119 density with sodium diatrizoate to 1.125 g/ml and increase centrifugation time. Viability of granulocytes has not changed. And the amount of isolated granulocytes increased too, while RBC sank to the bottom as expected. But it is "off lable" usage of gradient, maybe you found another solution?
Best regards!
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