Trypan blue fluorescence staining
Harbor Branch Oceanographic Institution (harbolon@CLASS.ORG)
Fri, 25 Mar 1994 08:10:10 -0800 (PST)
For everyone who is interested in the trypan blue
staining/fluorescence..here is the info. I use the standard viability
Trypan blue staining solution put out by GIBCO which contains a 0.4%
solution of Trypan blue in 0.85% saline. For one million cells, I add
a volume of the solution which results in a 1:10 final dilution (i.e. for
1.0 ml of cells I add 0.1 ml of the stain to the tube). Let the tube
incubate at room temp for about 10 minutes, then look at it on the flow
in the red region (675) and collect histogram data on log scale. You
should observe two peaks, the magnitude of each depending on the
viability of the culture. The first peak (dimly fluorescent) will be the
viable cells which, if you looked at them on the fluorescence microscope,
will have a thin red fluoresent ring around the cell. The second,
brighter peak, are the dead cells which are unable to "exclude" the dye
and thus are observed as bright red fluorescing cells. I have used a
number of cell types with various degrees of "viabilities" and one can
easily observe the two peaks shifting up and down depending on the
viabilities of each cell preparation analyzed. There was a mention of
serum absorption of the dye--I haven't observed any serum effects at the
concentration used--. Hope this is a usefull technique. I will be
submitting a paper to Cytometry shortly. Have fun!!
--Ross--
Ross E. Longley, Ph.D. * Harbor Branch Oceanographic Inst., Inc.
Immunology Group Leader * 5600 U.S. Highway #1, North
Div. Biomed. Marine Res. * Fort Pierce, FL 34946
Phone (407) 465-2400, 486
FAX (407) 465-1523
e-Mail harbolon@class.org
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