aquilar

Thanks Tr0x. I think you're right: the top layer is the lysate (cytoplasm etc). The middle layer may be RBC membranes (?) or clotting factors, as you say. I'm using bird blood, so the RBC are nucleated. Maybe the middle layer is DNA.

Hi guys. I have a quick question about blood lysis, and I hope you can give me some pointers. I am doing a lab technique which requires erythrocyte (red blood cell) lysate. I collect whole blood (using heparin), centrifuge and discard the plasma and white blood cells. The red blood cells are then added to 4X their volume of distilled water and put on ice for 5 minutes ('lysis stage'). It is then centrifuged again, yielding this: (sorry the image isn't great quality) There are three layers: - a dark red pellet at the bottom [blue line] - a cloudy middle layer, very gloopy/gelatinous [pink line] - a clear pink liquid on top [yellow line] My question is: which of these layers is the 'red blood cell lysate' ? I've been told to use the supernatant, but again I'm not confident which layer this refers to. Clearly it is not the pellet, but is it the liquid at the top, or the cloudy gloopy layer in the middle? Any thoughts would be very welcome! Thanks a million