Stoyan, As you note, the frequency of antigen specific T-cells is quite small. Earlier this year we reported the a multiparameter method to detect such cells. (Mathioudakis G, Coder D, Fefer A. Multiparameter cytokine-specific affinity matrix assay for the determination of frequencies and phenotype of antigen-reactive T cells. J Immunol Methods. 2002 Feb 1;260(1-2):37-42.) This uses the affinity matrix for cytokine capture in conjunction with cell surface and viability markers to distinguish live T-cells activated in response to a selected antigen (CD4,8,69; TO-PRO-3 for viability.) The number of viable T-cells secreting either IFN-gamma or IL-4 can be determined simultaneously. As the frequency such cells is small, and you would like to detect perhaps a thousand cytokine secreting CD4 cells, you need to screen around 6 million cells. If your cytometer can analyze around 5000 events per second, it would take about 20 minutes per sample (set the threshold carefully to eliminate debris.) Of course, faster (while still analyzing single cells) is better. Dave ----------- David M. Coder, Ph.D. Consultant in Cytometry Seattle Washington tel. 206-499-3446 email: d_coder@msn.com -----Original Message----- From: Stoyan Dimitrov [mailto:dimitrov@kfg.mu-luebeck.de] Sent: Wednesday, October 30, 2002 2:31 AM To: cyto-inbox Subject: antigen specific T cells Dear colleagues, I have the following problem. I need to detect very small quantity antigen specific T- cells (ASC). After vaccination with hepatitis A. For this reason I just cant use MHC tetramer technology and BD FastImmune Antigen-Specific Cytokine Detection. The only possibilities that I have are ELISPOT and Cytokine Secretion Assay- Cell Enrichment and Detection Kit (PE*) MACS. The both are very sensitive but the second also is muiltiparametric. So I can detect ASC on different T population on Cytometer (I mean simultaneous detection of cytokine production and surface markers) I looked at this method very carefully and I found a problem. Is a method representative? I mean the following. The method is based on enrichment of ASC. Before enrichment with a magnet, anti IFN g positive cells are 0.247 % from CD4 population. After enrichment you have 1292 IFN-+ CD4+ T cells per 106 total CD4+ T cells. That is 0.1292 %. So you lose approximately half of the antigen specific cells. See for example: http://www.miltenyibiotec.com/index.php?site=products-cytokine And after that click on Cell Enrichment and Detection Kit (PE*) for IFN G. In case you have very small quantity of ASC and is not possible to detect them before enrichment how can be sure that you can get their exact number after enrichment. See Cell Enrichment and Detection Kit (PE*) for IL4 for example. Thank you in advance for your support. I must decide the right method for detection of my little population. Do you know if there is protein extract (or better- peptide mixture to get T cell response) from Hepatitis A virus? Do you think ELISPOT is better for detection of such a cells? I really would appreciate any help. Stoyan Dimitrov Klinische Forschergruppe Neuroendokrinologie Medizinische Universitdt L|beck Ratzeburger Allee 160 Haus 23a 23538 L|beck Tel.: +49 (451) 500-3644 Fax: +49 (451) 500-3640
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