From: Howard Shapiro (hms@shapirolab.com)
Date: Mon Feb 09 1998 - 18:59:58 EST
Alice Givan asks: >Can someone point me to the state-of-the-art for cell cycle analysis with: >1) cells fixed in formaldehyde >2) live cells > >We have GFP-fusion-protein-transfected cells and, in the first instance, need >to fix in formaldehyde (not ethanol) to retain the GFP but want to get good DNA >profiles. Is there a stain that works better than PI with formaldehyde-fixed >cells or is there a method for opening up the fixed histones/DNA so as to >improve the PI profile without losing the GFP? > >In the second instance, we would like to keep the cells alive during this >procedure. Is there a staining method with one of the Hoechst dyes that will >give us histograms sharp enough to allow cell cycle analysis or is there any >better vital alternative? > Hoechst 33342 (HO342) can work well with both formalin-fixed and unfixed cells. We find that addition of 0.1% Triton X-100 improves resolution of DNA histograms on formaldehyde-fixed cells stained with 1 ug/ml HO342. Gentler permeabilization with saponin or Tween can also be used if Triton is a problem with GFP. We have also recently noted that leaving cells in formaldehyde for long periods of time (days) greatly increases background fluorescence in the FITC channel; this seems to be due to a chemical reaction between HO342 and reaction products of formal- dehyde. We have had some success in decreasing background by adding glycine, a procedure suggested by Richard Riese. Good vital staining with Hoechst depends, first, on using a relatively high concentration of the dye, and, second, on neutralizing efflux pumps if the cells have them. Awtar Krishan described the use of trifluoperazine and verapamil for blocking the pump; cyanine dyes appear to do the same, as noted by Crissman et al at Los Alamos. We can generally get CV's below 5% on live lymphoblastoid cells with HO342, and have gotten as low as 2-2.5% in cells simultaneously stained with HO342 and DiIC1(5) (this was demonstrated at the Portsmouth, NH SAC meeting in 1981, when we brought a dual-beam Cytomutt up from Boston). -Howard
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