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PBMC and granulocyte isolation from human whole blood - (Feb/17/2014 )

Hi all,


I have been using histopaque very unsuccessfully to isolate granulocytes and PBMCs from human whole blood. I tried layering histo-1077 onto histo-1119 followed by the blood. I did not get a clear distinction of layers (all at room temp). Second I tried doing an initial layering on histo-1119 to remove RBCs (worked lovely) followed by an additional layering on 1077. Although the leukocyte layer on the 1077 looked good, after spinning the 1077 seemed diffuse throughout the sample and there were no clear layers.


Any advice or thoughts would be greatly appreciated.


Thank you. 


We do it here for PBMCs, granulocytes and originally other fractions too, but I need to ask someone about specific type of histo used. But there definitelly was only one histopaque type and one layering.


But as I remember, the histo with PBMCs doesn't look distinctly clear with the monoclear "fuzzy ring" in it. Only after you take them out, the layers become clearly separated.


Perhaps it would be a good idea anyway to post here the whole exact protocol you are using, starting with the anticoagulant used for blood, age of the sample etc. so we can see more details.