Re: simultaneous surface/DNA analysis
Piet van Erp (P.vanErp@DERMA.AZN.NL)
Thu, 27 Apr 1995 08:54:28 +0100 (MET)
>
>We are currently seeking a protocol which will allow us to do three color
>surface immunophenotyping together with DNA content analysis. We can
>already do the simultaneous surface staining/DNA staining quite nicely, but
>only in two-color surface combinations. We have a FACStarPlus with dual
>lasers
>currently set at 488 and 600 nanometers. Since we routinely need this
>configuration for FITC, PE, APC, and Texas-Red in various combinations for cell
>sorting, it would really be best if we didn't have to swap out the dye head to
>use UV (for Hoescht, etc.). In other words, we'd really like to find a
>fluorochrome combination which will work using the existing beams. We've tried
>FITC and PE (laser 1) with Texas-Red and 7-AAD (laser 2), but can't seem to
>find a working combination of voltages and compensations on the latter. Since
>7-AAD is very bright at 600nm excitation, we can't put this on laser 1 because
>we still can't compensate it out of the Texas-Red due to the software
>limitations. My understanding and previous experience suggests that we'll have
>similar problems using PI. Any suggestions would be much appreciated.
>
>
For simultaneous staining of two cytoplasmic antigens and DNA we use FITC,
PE and To-Pro-3 (TP3). We have an Elite and use two air-cooled lasers, Ar488
en HeNe635. We changed from PI to TP3 because simultaneous PE/PI
measurements need so much compensation that we didn't trust our DNA
histogram calculations anymore. Using TP3 no compensation is needed
(emission of TP3 peaks is above 640 and peaks at 661) and there are no
problems with DNA histograms. However, with three color surface
immunophenotyping in combination with DNA content analysis, compensation
between Texas Red and TP3 will certainly be necessary.
Am I right when I worry about the shapes of DNA histograms after electronic
compensation of overlapping fluorescence spectra? How does cell cycle
software such as ModFit and MultiCycle handle DNA histograms with 70% or
more compensation as with the PE/PI combination?
-----------------------------------------
Piet EJ van Erp
University Hospital Nijmegen
dept. of Dermatology
P.O. Box 9101
6500 HB NIJMEGEN / The Netherlands
E-mail: P.vanErp@derma.azn.nl
Voice: +80-613548
Fax: +80-541184
-----------------------------------------
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