Hello again, Thanks to everyone for all your help. I've copied the replies below for anyone else that may be interested. 1) See the CDC MMWR from 1995: ftp://ftp.cdcnpin.org/MMWR/1994 select HIV Compilation94.pdf Use universal precautions, bleach and formaldehyde. 2) I would recommend 2-5% FRESH PFA for 8 hrs. See: Recovery of infectious human immunodeficiency virus from cells treated with 1% paraformaldehyde. Carol H. Aloisio and Janet K. A. Nicholson. J. of Immunological Methods 128 (1990) 281-285. 3) We have used 1% paraformaldehyde for 1 hr. for several years with no problems. Inactivation of HIV-1 has been confirmed. 4) We use the standard BD staining/lysing/fixing procedure and regular precautions (gloves, clean instrument w/bleach). This has been studied and published by Janet Nicholson, PhD, Division of HIV/AIDS 6) We have been using 2% paraformaldehyde (in PBS) for fixing the stained cells. I do not know where we got this protocol. Nevertheless, I did testing on infectivity of post-fixed cells. The infectivity is almost gone even after a few minutes when the cells (H9/NL4-3) is fixed in 2% paraformaldehyde. 7) Paraformaldehyde, at 0.5% to 1%, is the standard fixative for cells to be analyzed for flow cytometry. It will unquestionably inactivate HIV which is very sensitive to chemical treatment. I'm sure you can obtain a protocol from Becton-Dickison or any manufacturer of clinical based flow cytometers. 8) We have always fixed cells in 4% paraformaldehyde overnight before flow. I know that the fixation works after a shorter period of time, but so much of our work was labor-intensive staining, that we would be happy to let the stained cells sit overnight at 4 degrees inside aluminum foil (light protected) before running them the 5) It is best not to chemically treat the specimen [ fix ] prior to labeling with Abs since a decrease in fluorescence intensity may occur. Some success has been had with simultaneous stain and fix [paraformaldehyde]. next morning. 9) Standard formalin/paraformaldehyde fixation for 1 hour seems to be enough to inactivate the HIV virus. See: Lifson, J.D., Sasaki, D.T., and Engleman, E.G., Utility of formaldehyde fixation for flow cytometry and inactivation of AIDS associated retroviruses. J. Immuno. Meth. (1986) 86, 143. There are several recent ones from the CDC as well but essentially variations of the same theme. Thankyou again to everyone who sent all this information, it will be very useful. Laura.
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