In response to several of the replies I've gotten: 1) According to Pharmingen, the Harvard x-reactive antibody website, and Picker's paper the antibody is cross-reactive. 2) I've always staining before fixation with CD69 and other surface markers, on the now-circumspect rationale that CD69 was a surface marker! (I don't mean to imply that CD69 is not on the surface but that my logic was questionable). 3) Yes, I do understand that CD69 expression is transient, lasting for only a short period. But with samples taken daily over 7-10 days you'd think you'd see something! For venezuelan equine encephalitis in particular since the animals survive and resolve the infection fairly quickly. 4) Our problems with CD69 extend into the realm of cytokine staining after stimulation as well as the virus-infected animals. But after reading the replies it really looks to me that CD69 must be shed very quickly from the surface unless fixed in the golgi by brefeldin A. So I think that looking for cells responding to virus CD69 is probably just not a good marker to look at while for ICS it will work, if we do the staining after brefeldin A & fixation. Douglas S. Reed, Ph.D. Microbiologist Respiratory & Mucosal Immunity Department of Aerobiology & Product Evaluation Division of Toxinology & Aerobiology U.S. Army Medical Research Institute of Infectious Diseases 1425 Porter Street, Fort Detrick Frederick, MD 21702-5011 301-619-6728 301-619-6911 fax doug.reed@det.amedd.army.mil
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