From: Joe_Trotter@BD.com
Date: Wed Sep 17 2003 - 20:03:38 EST
Hi Yoav,
Yes, I have a good idea why there is no decrease in blue, it cannot
make it to and through your 424/44 filter. Your 510SP is reflecting light >
510 nm and there is nothing for the 424 filter (which is too short a
wavelength for blue). Bill Telford has a nice web page at
http://home.ncifcrf.gov/ccr/flowcore/filters.htm where you can see many of
the different filters and their uses. You will probably want a 460 LP to
pass blue and reflect into the violet (405/20). A 485/22 for blue will also
pick up scattered 488 nm laser light if you are not careful (you need to
align precisely and stop down the iris a little for the UV laser detectors
to exclude as much 488 nm light as you can). There are several ways to
minimize high blue background from a 488 nm laser. Many people have
actually used a 530/30 filter for "blue", or even custom filters that
collect light from > 488 nm to around 530 nm to avoid 488 light getting
into the "blue" detector. If you have a tunable primary laser and do not
need 488 nm excitation , then another way is to use 514 nm for your primary
laser, put 530/30 nm filters in for FSC & SSC (514 nm gets in just fine),
and then use the standard 405/20 (violet) and 485/22 (blue) with a 460
dichroic for good Indo-1 measurements using UV excitation.
Best,
Joe
Yoav Altman
<yoav@pobox.c To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
om> cc:
Subject: Ca++ flux problem
09/16/03
07:15 PM
Flowers,
An investigator bringing me samples for calcium flux studies has some
questions about her data that I'm not able to answer. I'd appreciate any
help from the calcium wizards out there.
We're running Jurkat cells loaded with Indo-1 on a FACSVantageSE DiVa. I'm
using 80mW of MLUV from a Krypton laser for excitation and BD's 510SP to
split the signal to my two UV detectors. UV1 and UV2 have 405/20 and
424/44 filters respectively; nothing unusual, as this all came from BD's
calcium filter set. Voltages for the PMT's are 580 (UV1) and 520 (UV2).
When we stimulate the cells with OKT3 we see a nice increase in violet
(Calcium bound Indo-1) emission. The problem is that we don't see a
corresponding decrease in blue (unbound) fluorescence. Do any of you have
any ideas as to why we aren't seeing a decrease in unbound Indo-1
fluorescence?
Thanks,
Yoav
Dot plots of cells before and after stimulation with OKT3.
[IMAGE]
Time-course of the same data set.
[IMAGE]
[IMAGE]
--
Yoav Altman
Manager, High Throughput Cell Analysis Core
The BURNHAM INSTITUTE
10901 North Torrey Pines Road
La Jolla, CA 92037
Voice: (858) 646-3100 xFLOW (x3569)
Fax: (858) 646-3167
email: <yoav@burnham.org>
(See attached file: P4AED6998)
(See attached file: P4AED6998_1)
(See attached file: P4AED6998_2)
(See attached file: P4AED6998_3)
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