centrifugation of fresh hemocytes - (Jan/27/2014 )
A simple question, but i'm fed up with this.
I work with hemocytes of invertebrates. Many articles perform centrifugation steps after collecting the hemolymph, whether in eppenddorf, whether directly on a 96-well plate. Theses cells are circulating blood cells but display quite good adherence.
Here is my problem: after centrifugation ( I tried 5 min at 300g, 10 min at 300g and 5 min at 1000g, in eppendorf and with 96-well plates, flat-bottom and round bottom). I took the supernatant off and tried to resuspend the cells with PBS and then performed my classical viability protocol for flow cytometry analysis… But the cells have gone… These velocities are the one used in articles… and i can't increase the velocity of centrifugation because i want my cells in viable status... I can't figure out where my trouble comes from.
Apparently it is not enough to have pellet (i don't visually see it by the way :S) that will not detach after a PBS-washing step … but many articles do not report that, so the problem is from my protocol.
Many thanks in advance,
I can't comment specifically on hemocytes, never having worked with them, but in general for cells, increasing RCF (g) is not usually useful, as it tends to damage the cells more. I would try 100 RCF for 5-10 minutes and work from there. I would be surprised if you can't see a pellet, most cell pellets are usually quite visible, but this does depend on the number of cells collected.
Thank you for responding,
I know it is weird not to see pellet, although there must be enough cells (approximatively 1.10^4 cells in total per well) but still it is not a lot. I is more suitable for me to work with such concentrations because they are the physiological ones.
Maybe i should centrifuge primarily in the collecting 10 ml tube containing all the cells that i will seed later, resuspend the cells with less volume so that i concentrate the cells, and then seed them on my 96-well plate...
Anyway, this does not explain why after centrifugation, when i discard the supernatant and resuspend with PBS, i can't see any cells remaining… Even with small number of cells, at 400 g for 5 min, the cells should stick to the bottom and not go out when taking off the supernant and performing washing step, isn't it??
Where the cell pellet would be on a specific tube, depends on the tube, the size of the cells and the type of rotor used for the centrifugation - if using a fixed angle rotor then the cell pellet should be on the wall of the tube. Swinging bucket rotors should result in the cells being pelleted at the bottom of the tube, but if the cells are small, they may smear up the side of the tube under higher RCF. That's why I was recommending lower RCFs.
Thank you for responding,
I tried again at 300 g for 5 min, on a 96 well plate and this time i was able to quantify cells after discarding the supernatant and resuspending the cells… But i was not able to see where the pellet stuck… not with the eye's acuity at least… But i am still doubtful regarding the technique and how this does not provoke stress to the cells and which fraction i loose when i perform washing steps (smaller cells or cells damaged that are not well pelleted may be washed and the final resuspension would contain only the bigger and most resistant cells) ...
Centrifugation at high RCF provides a lot of stress too - imagine being squashed to the walls of a tube at several hundred times the force of gravity...