From: Ray Hicks (rh208@cus.cam.ac.uk)
Date: Tue Mar 10 1998 - 12:36:39 EST
Hi Bill,
Have you tried using a filter that's better matched to red 613's emission
peak (eg 630 bp (or better still 610), with your red laser switched off, if
necessary?).
As an aside, but possibly relevant; does anyone know which wavelengths
actually get passed by the filters in the FL3 position on benchtop FACS
machines? My FACSort has a 650LP in the FL3 position, mounted at 90° to
the light path, aren't these things usually designed for use at 45 degrees?
If it is meant to be used at 45 then it won't be splitting the light as
claimed. Sadly I haven't got a scanning spectrophotometer, but someone who
has could maybe pop the filter out and give it a go, (holding the filter in
the cuvette slot at 90 degrees to the illumination path), I'd be interested
to get a copy of the results.
Ray
At 7:05 -0800 9/3/98, william nostrom wrote:
>Hi all.
> Last week I tried running an experiment using red613 and I wound up
>with very confusing results. I was trying to sort stem cells on a
>FACStar+. Our fluorochromes were: Sca-1 FITC, Lineage PE, CD34 REd 613
>and c-kit APC. We also threw in PI to gate out dead cells. I ran into
>trouble when I was trying to set up my compensation. Everything looked
>great until I put the red613 on, and that looked terrible. I am using a
>675/20 filter on FL3, and it seems to totally absorb all of the signal
>that should have been seen. Looking at an FL2 vs FL3 dot plot, on just
>my 613 positive control tube, I had a good negative group, and my PI
>dead cells, and that was it. (I back gated on the PI cells to see where
>they fell on a FSCvSSC, that is how I know they were dead.) Now, before
>you say my staining was bad, I took the tube and ran it on our
>FACSCalibur and it looked perfect. the PI cells were in both FL2 and FL3
>channels and there was a very bright FL3 signal that had to have been
>the 613 cells. So my question is what is the difference between the
>Calibur and the Star? The Calibur uses a 670 LP filter on FL3. I checked
>my fluidics and rechecked alignment with CRBC's. Does anyone have any
>suggestions?
>TIA
>Bill Nostrom
>Trudeau Institute
>Saranac Lake, NY
>
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Ray Hicks
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