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Kevin Holmes, Ph.D.
Head, Flow Cytometry Unit
Office of the Scientific Director
National Institute of Allergy and Infectious Diseases
National Institutes of Health
Bethesda, MD
>----------
>From: dehman@hookup.net[SMTP:dehman@hookup.net]
>Sent: Tuesday, July 02, 1996 8:50PM
>To: Cytometry Mailing List
>Subject: Excitation of APC by UV(?)
>
>
>I know, what does that subject heading say?
>That's just to catch your attention. I am hoping for some help with a
>tricky sorting setup. A premier customer of ours wants to do
>five-colour
>immunofl., using FITC, PE, TxR-PE, APC and AMCA or Cascade Blue. We are
>seeing something odd in FL4 and FL5. Here are some facts.
>
>FACStar Plus with active 7th detector option (to enable 5 colours)
>three lasers: I-70 running 488 at 200 mw, I-90 running UV at 35 mw, SP
>HeNe
>at 35 mw. I-70 is laser one. UV and HeNe are collinear, at ~20
>microsecond
>delay.
>Iris reduced to minimum possible (about half closed) in FL4 channel.
>Pinhole in place in front of APC PMT.
>Filters: 640 LP dichroic, 670/20 BP and a GG420LP (absorption filter)
>in
>front of APC PMT and a DF424 in front of AMCA PMT.
>Here comes the puzzle:
>I did not think that APC could be excited by UV. But we see a signal in
>the
>APC channel from FCSC APC beads when only the UV laser is unblocked. It
>is
>10% the intensity seen from the same beads when only the HeNe laser is
>unblocked. (In both cases, the 488 beam is unblocked.) I thought that
>we
>were seeing light scatter from the UV laser that was penetrating the
>640
>and the 670, so that is why the 420 is there as well. Use of the 420
>made
>no difference other than reducing all signals by about 20%. So now I
>seem
>to be seeing true red fluorescence of APC excited by UV illumination.
>My
>paradigm shifts! My flow cytometric bubble bursts! (I don't know so
>much
>after all!!!)
>
>Seriously, does anyone have any ideas as to whether APC really could be
>doing this. Molecular Probes can't tell me for sure. (They are "too
>short
>on resources" to do a spectrofluorometer run to check) Or more likely,
>what am I missing here in instrument setup?
>
>Thanks very much.
>
>
>
>_______________________________________
>Dave Ehman
>Technical Applications Guy
>BD Canada
>PH: (800)267-5586 Xt. 2004
>Fax: (613)836-3580
>Email: dehman@hookup.net
>_______________________________________
>
>
>
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