Hi Heather, A quick and fairly easy method is to monitor cytokine (eg IL2 or IFN-gamma) production. You can use brefeldin A (available from Molecular Probes amongst others) to make the cells accumulate cytokines internally, then fix, permeabilise and stain for the cytokine of your choice, this can all be done in a day but presumes that the cells producing the cytokine would go on to proliferate. B-D provide an html protocol and background pdfs at their web site: http://www.bdfacs.com/source_book/html/23_3240.shtml Alternatively for a longer term assay you could follow actual cell division and proliferation using CFSE (carboxy fluorescein succinimdyl ester - also from molecular probes) or one of the PKH dyes available from Sigma, with these dyes, the fluorescence diminishes as cells divide, so you can see the daughter cells emerging as a dimmer fluorescent peak. Working out how many cells were initially stimulated is a bit tricky since you need to be able to work back from the number of daughter and granddaughter cells, and would be less reliable for lower frequencies of responding cells - luckily Alice Givan has published a model for the calculation ( A.L. Givan et al, J. Immunol. Methods, 1999, 230:99-112 - there is also a correction published in the next issue poinng out a typo in the equation where the dividing sign was missed out between the two bracketed components), and this has been incorporated into ModFit. Ray > From: "Heather Medbury" <Heather_Medbury@wsahs.nsw.gov.au> > Date: Mon, 06 Nov 2000 16:31:37 +1100 > To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> > 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. > > >
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:37 EST