Hello Yu-Min What you are seeing is activation induced CD4 downregulation. To circumvent this for subset marker detection, I label the cells prior to stimulation with the antibody (azide free)at RT then perform stimulation as normal. If doing this technique its better to use monensin as a protein transport inhibitor. Another trick is to use slightly sub-optimal doese of PMA etc). I find this can reduce the amount of CD4 downregulation whilst still maintaining good cytokine expression. You didn't say what type of CD4 cells you were looking at - for human PBMC certainly, cytokines are easily detectable at a later window of 24hr. By this time, cells have always (?)recovered detectable CD4 expression. Contact me if you ned more info Gill Webster PhD Genesis Research and Development PO Box 50 Auckland email: g.webster@genesis.co.nz direct dial 00 61 9 3743708 fax: 00 61 9 373 2189 -----Original Message----- From: Yu-Min Huang [mailto:yu-min.huang@neurotec.ki.se] Sent: Tuesday, July 10, 2001 11:53 PM To: cyto-inbox Subject: how to measure cytokine producing CD4 cells Dear Flowers: We have a problem to measure cytokine producing CD4 cells. After stimulation with PHA or PWA with ionomycin, it is hardly to detect CD4 cells. Any one has good resolutions for this problem regarding stimulies and incubation time (we use PWA + Ionomycin for 6 hours and monocin for the last two hours)? Best regards, Yu-Min Huang Division of Neurology Karolinska Institute Huddinge University Hospital 141 86 Stockholm Sweden Tel: +46 8 58582274 Fax: +46 8 58582270
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