Thanks to all that responded to the following query: >Dear Purdue site: > >Currently we use a 1:4 dilution of methanol free, EM grade, 10% >formalin in PBS as a fixative for immunophenotyping of human blood. >We have some preliminary evidence that this concentration is >contributing to a decrease in fluorescence for CD45RA Fitc when >analyzed at 24 hours. > >A literature review indicates this is a stronger concentration than >the norm (0.5, 1.0 or 2%). Our specimens are processed with either >FACS Lysis or Coulter Immunoprep, then fixed. Any opinions or >suggestions concerning what concentration best preserves as well as >inactivates viral components? In summary, 1 to 2% paraformaldehyde or formaldehyde works well for routine CD4/CD8 immunophenotyping and is recommended by the CDC. Many other antigens hold up well with these concentrations. However CD45RA, used in analysis of naive T cell subsets, decreases in fluorescence over a 24 hour period and lower concentrations of fixative are necessary for this, and possibly other less commonly used, antigens. Results from the literature: MMWR, May 08,1992/41 (RR-8);001: Guidelines for the Performance of CD4+ T Cell determinations in persons with Human Immunodeficiency Virus Infection, pg 6 of 16: iii. Incubate all tubes in the dark during the immunophenotyping procedure. j. Before analysis on the flow cytometer, fix all sample tubes after staining and lysing with 1%-2% buffered paraformaldehyde or formaldehyde. (This should be done even if the commercial lysing/fixing reagents contain a fixative). "Utility of formaldehyde fixation for flow cytometry and inactivation of the AIDS associated retrovirus." J Immunol Methods, 1986, Jan 22;86(1) 143-9, Lifson JD, Sasaki DT, Engleman EG. We found that both formaldehyde (0.37% v/v) and paraformaldehyde 0.5% w/v) completely inactivated the infectious activity of both free and cell-associated lymphadenopathy associated virus. "Effects of cellular fixatives on human immunodeficiency virus production." Cytometry. 1990;11(5):647-51, Cory JM, Rapp F, Ohlsson-Wilhelm BM. Thirty minute fixation with formaldehyde (1.85%), methanol (absolute), methanol:acetone (1:1), or paraformaldehde (0.5%) reduced the infectivity of HIV-1 infected H9 cells by greater than 99.99%. Responses from the "flowers": From: "Donnenberg, Albert" <donnenbergad@MSX.UPMC.EDU> Date: Sat, 16 Feb 2002 17:00:41 -0500 We made the same observation some years ago. Of dozens of specificities tested, CD45RA was the only one affected in this concentration range. We now routinely use a final concentration 0.25% methanol-free formaldehyde. This is sufficient to fix the cells (but just barely). Albert Donnenberg University of Pittsburgh If you are using the FACS Lyse from BD, it has formaldehyde in it. This already fixes the cells, so I would recommend a very low concentration (<1%) of a final step paraformaldehyde fix. Randy T. Fischer NIH/NIAMS Building 10, Room 6D57 9000 Rockville Pike Bethesda, MD 20892 (301) 594-3537 fischer1@mail.nih.gov From: "Mark Kukuruga" <kukuru@med.umich.edu> It's not so much the concentration as . . . Both Immunoprep (the kit?) and FACS Lysing Solution include a fixative. Therefore, you're fixing your samples twice. The consequence of this could be reduction in fluorescence, or more likely an increase in background noise - - either way, you lose resolution. MAK. From: "Simon Monard" <smonard@trudeauinstitute.org> The cells are fixed after FACS Lysis treatment, you could use a lower concentration of formaldehyde, 0.5 or 1%. You can also wash the fixative out after an hour or two and suspend your cells in PBS, the cells will remain in pretty good shape for a few days or longer. Often after formaldehyde fixation the autofluorescence of all cells increases gradually, washing the fixative out prevents this. Simon Monard FACS Lab Manager Trudeau Institute Saranac Lake NY12983 Thanks for these and other responses along the same lines. Sincerely, Lorrie Epling
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