Hi Heather For what its worth, we've been able to detect significant numbers (5%+ initially identified using tetramers) of memory cells that produce cytokines when stimulated with autologous PBMCS presenting relevant viral peptides. However, we've found that you need to add Brefeldin A (or monensin) fairly quickly (after 60 - 90 minutes) as we suspect T-T killing wiped out the potential responders when incubated overnight before adding a secretion inhibitor. Our results to date to detect activation of naive cells responding to allostimulation using intracellular cytokine staining aren't as clear cut. If you (or anyone else out there) has any suggestions re allostimulation I'd appreciate them. Our ICS procedures are based on the methods outlined in the following papers: Jung et al, 1993 Detection of Intracellular cytokines by Flow cytometry, J. Immunol. Meth. 159, 197. Prussin et al, 1995 Detection of intracytoplasmic cytokines using flow cytometry and directly conjugated anti-cytokine antibodies, J. Immunol. Meth. 188, 117. Suni et al, 1998 Detection of antigen-specific T cell cytokine expression in whole blood by flow cytometry J. Immunol. Meth. 212, 89 Good Luck Ian Nicholson Melbourne, Australia. > -----Original Message----- > From: Heather Medbury [SMTP:Heather_Medbury@wsahs.nsw.gov.au] > Sent: Monday, 6 November 2000 16:32 > To: Cytometry Mailing List > Subject: lymphocyte reaction to antigen in vitro > > > Hi all > > Not strictly a flow question, but I plan on using flow to examine the > cellular response > > I wanted to look at the human immune response to particular antigens. > Iwanted to add > antgien to human leukocytes in culture and detect the immune response by > flow and > proliferation assays. > I realise however that the immune response will involve a very small > number of T cells > specific for the antigens, so I probably wouldnt be able to detect the > response. Though > I would look by flow for the activated T cells and examine them only. > > Any hints, comments or suggestions on whether it will work or how I can > get it to work. > Any references you know of that may be useful > > Thanks > > Heather Medbury > Sydney Aus. >
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