Setting up indo-1

vanburen%flovax.dnet@rocdec.roc.wayne.edu vanburen%flovax.dnet at rocdec.roc.wayne.edu
Thu Apr 13 10:13:04 EST 1995


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 at rocdec.roc.wayne.edu
\ \ \   Immunology & Microbiology
 \_^_/  Wayne State University, Detroit, Michigan, USA



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