EGFP

ar3@mrc-lmb.cam.ac.uk ar3 at mrc-lmb.cam.ac.uk
Tue Aug 25 05:04:45 EST 1998


>Date: Mon, 24 Aug 1998 10:04:06 -0500
>From: "Jacqueline Saleh" <jacqueline.saleh at ariad.com>
>To: Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
>Subject: EGFP
>
>
>Dear Flow-ers:
>
>We are working quite a lot with EGFP and are seeing quite a discrepency in
>results
>between what we see under the scope and the fluorescence readout on the
>instrument.
>I looked at two samples under the fluorescent scope and the difference in
>fluorescence is
>quite significant. Cells are then trypsinized, washed and read using the
>FacsCalibur and
>the results of these two samples are quite similar. I thought maybe the
>trypsin might be
>pulling off the EGFP but EGFP is internal. Does anyone know what might be
>going
>on here????? Thanks in advance for all your help.
>
>Jackie Saleh
>Ariad Pharmaceuticals
>saleh at ariad.com


Hi Jackie,

You need to look at the filter sets you are using. I don't know what filter set 
is in your scope, but if you are using the standard filter set in the Calibur
then you will be missing the peak emission intensity for EGFP. The Calibur green
filter on FL1 is a band pass (BP) centred on 530nm with a range between 515 and
545. EGFP emits maximally at 510nm and tails off sharply.

If I am analysing EGFP on the Calibur, I use a 510nm BP with a range between
approx. 505nm to 515nm. I got this filter from Glen Spectra in the UK (see
below). You will need to get them to cut it to size to fit the Calibur.

Hope this helps.

Andy.




Glen Spectra
2-4 Wigton Gardens
Stanmore
Middlesex
HA7 1BG
UK.

tel: (0)181-204 5189
e-mail: gs at isa-gs.co.uk
Web: www.isa-gs.co.uk




Andy Riddell
PNAC
MRC-LMB
Hills Road
Cambridge
UK

tel (0) 1223 402218
fax (0) 1223 412178
email ar3 at mrc-lmb.cam.ac.uk



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