1. Follow the standard protocols published in methods books- for instance Current Protocols in Immunology. I am assuming that you are referring to human blood. Contamination of red cells stems from improper method of isolation on Ficoll gradients. Some lots of Ficoll may not be good. Lysing solutions will get rid of red cell contamination. You may also whole blood method for cytokine analysis. 2. At least some commercially available lysing solutions are also fixatives. They do permeabilize cells. So, it is suitable for subsequent intracellular cytokine staining. I have used BD lysing solution in conjunction with permeabilizing solution for intracellular cytokine staining of human peripheral blood T cells with good success (not an endorsement for BD product). 3. I am not familiar with cotton wool but nylon wool has been traditionally used to enrich T cells. Again, good starting point is any standard cellular immunology methods book-plenty of excellent books are available. 4. We routinely use normal ranges of PMA (~100ng/ml) and calcium ionophore such as ionomycin (1 mcg/ml) per one million T cells. You may want to titrate the best concentration for your experimental conditions. Good luck. S. Jayaraman, Ph.D. > -----Original Message----- > From: Rana Nagarkatti [SMTP:rana_nagarkatti@usa.net] > Sent: Wednesday, May 16, 2001 3:25 AM > To: Cytometry Mailing List > Subject: cord blood RBC > > > 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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