I am working with a fish and I want to measure the amount of DNA in the red blood cells using flow cytometry, to estimate the genome size. However, the blood from which i am trying to isolate the red blood cells from was not properly heparinized and was frozen in liquid nitrogen right after sampling and stayed in liquid nitrogen up to date. I have diluted the blood in PBS and centrifugate it at 4 degrees celsius, however, the pellet is a whitish fibrin-like clot that contains no red blood cells, and the supernatant contains no cells neither. With other samples that were properly heparinized the RBC are in the pellet. Where should I look for the red blood cells in my non-properly heparinized samples?
Has anything been added to the blood before freezing? The heparin does not effect the cells when frozen as far as I know.
Just chucking the whole blood in N2 will result in lysed cells.
first you wash the RBCs with glycerol three times than put into liquid N2 . You can store RBCs up to 9 years without any damage . When you want to use these RBC ,you wash the RBCs three times with isotonic saline and use these RBCs . If you directly put Whole blood or RBCs into liquid N2 than it will haemolysed .
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