>X-Sender: hmsmdpc@shell1.shore.net >Mime-Version: 1.0 >To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> >From: Howard Shapiro <hms@shapirolab.com> >Subject: Re: 7ADD vs Hoechst >Date: Sun, 23 May 1999 17:52:09 -0400 snip - Howard Shapiro writes: > >At 1 ug/ml, Hoechst 33342 gives good DNA histograms in fixed cells, >particularly when 0.1% Triton X-100 is added. However, I (and others) have >observed, but not formally published, that staining of formaldehyde-fixed >cells with Hoechst dyes and DAPI produces increases in fluorescence >background in the fluorescein and PE channels. We don't understand the >mechanism, but believe that the fluorescence is due to a chemical reaction >between the dye and formaldehyde or some product of formaldehyde. The >longer the time in fixative, the worse the background; once cells have been >adequately fixed, adding 0.15% glycine (a suggestion made to us by Richard >Riese) seems to neutralize the remaining fix and prevent further increases >in background fluorescence. If I weren't trying to use the formaldehyde to >neutralize HIV and other nasties in specimens, I'd be tempted to work with >unfixed or ethanol-fixed cells. > >-Howard > We have also witnessed that staining of formaldehyde-fixed DAPI produces increases in fluorescence background. This has been observed in experiments using human cells indirectly labeled with a Red-613 tagged antibody. The cells are permeabilized with tritonX and then fixed in 1% PF. We are looking at DNA using DAPI. In these experiments our FL3 channel (Red-613) appears to gain background fluorescence as we are running the cells through a Vantage. Amazingly, when the labeled cell suspension is first placed on the sample port, the cells exhibit normal looking, negative background fluorescence levels then, within a few seconds, all of the cells begin to show movement into the positive. The FL1 and FL2 channels do not show this fluorescence creep - and we cannot reproduce this phenomenon on a FACScan. It doesn't appear to be sample pressure related as this fluorescence increase happens in the same manner no matter what the psi of the sample differential. However, if one takes the cells off the sample port after they have migrated into the positive and then replaces them again - the background fluorescence initialy returns to normal negativity - but then soon begins it's slide towards the positive. There is a point where the migration into the positive appears to slow and stop - but it is typicaly a considerable distance from the baseline. This seems similar to what Howard has described - with the exception that we witness it in the FL3 channel, but NOT in the FL1 or Fl2. I will be very interested to see if the addition of the 0.15% glycine helps. Have any others seen this phenomenon as well? regards, barry <+>:<+>:<+>:<+>:<+>:<+>:<+>:<+>:<+>:<+>:<+>:<+> Barry Grimes FACS Laboratory Manager Hematopoiesis Center Univ. of Kentucky Medical Center Blood and Marrow Transplant Rm.cc418 Markey Cancer Center 800 Rose St, Lexington,KY 40536-0093 lab) 606-323-8193 fax) 606-257-7715 e-mail) bagrim1@pop.uky.edu <+>:<+>:<+>:<+>:<+>:<+>:<+>:<+>:<+>:<+>:<+>:<+>
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