Dear Flowers, Hi, We are using cord blood mononuclear cells for studying T Cell activation and we are facing some problems in the separation protocol using Ficoll-Hypaque. Based on literature it is clear that RBCs will come in the CBMC preparation but how to remove the contaminants is not clear. Lysing solutions are used but I am not sure if it works for intracellular staining, and also if it changes the stimulation pattern. Has any one tried these? Also can protocols such as using cotton wool coulmns help to purify the T and B cells. When using whole blood for intracellular staing it is suggested to use more of PMA and ca2+ ionophores is it because they are adsorbed or sequestered by the RBC's ??? So that the available levels can activate the T cells. And if this is true then lysing protocols may also be dogged by such problems as (atleast I) have not been able to get 100% RBC free pellets ( can someone please give a standard operating protocol for this method also currently I am using ammonium chloride method as given in the BD technical manual.)(also have seen others with such problems and will post the summary of all the responses). Thanks a ton (in advance) for all possible comments and hints. Regards, Rana Nagarkatti, SRF, Center for Biochemical Technology Mall Road, Delhi, India ____________________________________________________________________ Get free email and a permanent address at http://www.netaddress.com/?N=1
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