Problem with separation of lymphocytes - (Dec/24/2006 )
I am currently doing a project to observe the genotoxic effects of aspartame on lymphocytes by carrying out the micronucleus assay and sister chromatid exchange assay.I have carried out the normal protocol for culturing the lymphocytes.However I have had trouble harvesting the cells.After 72 hours of exposure to aspartame ,when I centrifuge the culture medium I fail to get a pellet of lymphocytes impeding the subsequent steps involving hypotonic treatment and staining with Giemsa.The protocol which I am currently following does not specify the details regarding centrifugation.So can you please suggest reasons for not getting a pellet or a protocol for harvesting the lymphocytes.
Maybe the reason for not getting a pellet is because your blood sample is not enough for culturing. Or another thing is how you proceed when separating lymphocytes from total blood - do you wait for the blood to decant or you separate with ficol?
Am not sure about ur experiment but just to pellet lymphocyte we centrifuge at 2000rpm for 5min. We get good pellets.