In article <2tie02INNi4k at medicine.wustl.edu>, HAVILAND at KIDS.WUSTL.EDU
(David L. Haviland, Ph.D.) wrote:
> Greetings one and all...
>> I need the advice of any other flow cytometry experts as well as those
> versed in immunoflourescence. We are trying to do some studies on primary
> fibroblast cell lines. Unfortunately we have had no success as human
> fibroblast are *hideously* autoflourescent. A negative control being
> simply just secondary Ab that is either FITC or PE tagged gives just a good
> a histogram as what should be a positive control. No other cell line or
> bulk cultres of cells give us this problem except for human fibroblasts.
> As far as we can tell (and my asking here a year or so ago) human
> fibroblasts are negative for Fc receptors.
>> Any thoughts would be appreciated!
If you have access to a flow cytometer with longer wavelength excitation
(dye laser or HeNe or Kr), then you might try seeing if the
autofluorescence is less; we've noticed this with numerous highly
autofluorescent lines (L cells come to mind as particularly bad). Dyes
such as Texas Red, Cy-5 and Allophycocyanin work for immunofluorescence at
these longer wavelengths. Another approach is to use the green
autofluorescence to "overcompensate" the orange channel, then stain with a
PE-reagent; there was a description of this technique back in the mid-80s
in Cytometry, I think. All this assumes that we are talking about true
autofluorescence - as I read your description, the unstained and 2nd step
alone histograms are equally bright. One last thought, if you anticipate a
very bright stain (ie, determinent is highly expressed), then simply lower
your PMT voltage or add neutral density filters in front of the PMT to
bring things back onscale.
Fox Chase Cancer Center