RE: Setting up indo-1
vanburen%flovax.dnet@rocdec.roc.wayne.edu
Thu, 13 Apr 95 11:13:04 -0400
Graham Smith (?) inquired about setting up Indo-1 on a FACS Vantage.
Several people have already posted useful instrument setups. Here, I post
yet another. But first, a little history. We originally developed an Indo-1
(and FITC and PE) protocol on an EPICS 753. Everything worked great, except
for the Indo-1. :) We were fortunate enough to be assisted by none other
than Carl June. With his expertise, he quickly located the problem (our
violet filter was too broad), and based on his advice we purchased a new
violet filter and haven't had any problems since. The same filters worked
fine on a FACStarPLUS, and now on our FACS Vantage. Without further ado,
here are the filters:
395 DF 10 (FL5)
525 BP (FL4) *
440 DC LP (to split FL4 and FL5)
All other filters are standard Vantage filters. *This originally came with
the EPICS; BD's 530 FITC filter should work just as well. Why fix something
that's not broken? (More sage advice from MAK.) :) The other two filters
were purchased from Omega (sorry, I can't remember what the designation was
for the 440 DC LP; something like 440DR?).
Also, I think everyone has pointed out that the dichroic mirror holder in
question (the one that "swings") is not appropriate for the Vantage (I
tried using a holder from a FACS 440 in the Vantage, before I found the
spare holders and mirror mounts in the "spares" box). Just a little bit of
rubber cement to mount the mirror on the mount, then screw it on to the
holder (look, no cardboard!). :) Use beads (or better yet, Indo-loaded
cells; are we analyzing beads or cells?) :) for the final tweak of the
dichroic to maximize FL5.
And now, a word about lasers. We're using our old Innova 90-5 argon ion
lasers; the primary laser tuned to 488 nm, 100 mW, and the second laser
tuned to all-lines UV, 200 mW.
Beads, beads, beads! To set up the FL5 detector, I like FCSC's Hoechst
beads, NOT the Indo beads (the Hoechst beads are brighter than the Indo
beads in the violet range). Spherotech's Rainbow beads are good for all-color
(multi-excitation, multi-fluorescence) instrument setup. I haven't tried
them with my Indo-1 setup, but I have a feeling the Rainbow beads will
replace the Hoechst beads.
/\/\/\_ Eric Van Buren, vanburen%flovax.dnet@rocdec.roc.wayne.edu
\ \ \ Immunology & Microbiology
\_^_/ Wayne State University, Detroit, Michigan, USA
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