From: ar3@mrc-lmb.cam.ac.uk
Date: Tue Aug 25 1998 - 05:04:45 EST
>Date: Mon, 24 Aug 1998 10:04:06 -0500 >From: "Jacqueline Saleh" <jacqueline.saleh@ariad.com> >To: Cytometry Mailing List <cytometry@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@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@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@mrc-lmb.cam.ac.uk
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