SP Cells

Ian Titley Ian.Titley at icr.ac.uk
Tue Nov 29 06:15:34 EST 2005


One of the original papers on this is Goodell et al J Exp Med 1996
1797-1806 who used 450/20BP, 675 LP and a 610 beam splitter (both
measurements, obviously, from the UV line) on a FACSStarplus.

We have used  a 424/44BP, 670LP and 610 beam splitter eg Jonkers et al
Stem Cells 2005 1059-1065 on a FACSVantageSE (we have also looked since
we had the DiVa upgrade and all is much the same, as one might expect).

So I suspect your blue filter and beam splitter should be OK (I can't
think that 525 or 610 beam splitter would make that much difference in
this assay?) but I would widen things out with a red long pass, 660/20
seems a bit restrictive. Look at both red and blue on linear scales and
the blue intensity should be in the ball park of what you would expect
for a Hoechst stained sample for DNA analysis (red voltage will probably
need to be set a little higher).

Good luck

Ian


Ian Titley PhD
Section of Haemato-oncology
Institute of Cancer Research
237 Fulham Road
London UK SW3 6JB

Tel +44 (0)20 7352 8133
Fax +44 (0)20 7352 3299

>>> Leonie Gaudry <L.gaudry at unsw.edu.au> 28/11/2005 05:42:54 >>>

Hi,
I am having a bit of trouble setting up the Voltages and Compensation
for
SP Cells.
I am acquiring the cells on a BD FACSVantage with DiVa option in
Digital Mode.
How critical are the filters,I have a Hoechst 33342 450/20 for the blue
and
a 660/20 for the red but I only have a 525DCSP for the splitter. Are
these
okay or not specific enough?

I also have a problem in the two papers I am referring too. One paper
states that the detectors should be in Linear mode while a second paper
is
in Log. Up till now I have been running in Log so which is correct and
I
would be very interested to hear anyones experiences.
Thank you for your help.
Best Wishes
Leonie





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