 |
 |
 |
 |
 |
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
>_______________________________________
>
>
>
|
|
|
|
|
|
CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web
|
|
|
|
|
|
CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web