Hi Claude,
I think it's going to be tough to see the Cy7 from the PharRed using
a -08 PMT. I'd recommend at least a -12, I'd prefer a -15.
Are you seeing the APC from the PharRed- you might see a bit
of that , which would verify that the Ab is working.
Also, you may squeak out a tiny bit more light by using a short
pass instead of going through a long pass to get to the Cy7.
Good luck
Peter Lopez
The Aaron Diamond AIDS Research Center
212.448.5188 (office)
212.448.5159 (fax)
212.448.5190 or 5110 (lab)
Claude Cantin
<cantinc@uhnres.ut To: mug@Cytomation.com,
oronto.ca> cytometry@flowcyt.cyto.purdue.edu
Sent by: cc:
owner-mug-cytomate Subject: no PharRed
@frii.net
05/23/2001 06:06
PM
Please respond to
cantinc
Hi All
I have been unable to detect a PharRed signal from samples I have been
given, and don't know if the problem is instrument or labeling error.
MoFlo set up is as follows:
Hammatsu 957-08 PMT all around
Omega Optical Filter Set XF501 (consists of 710 DRLP, 670DF40, and
740EFLP)
Melles Griot 35mW HeNe
Red laser alignment seems fine: "Molecular Probes AlignPlus beads for 633"
are almost off scale if no ND filter, at 399V, linear gain 1. BD's APC
like Calibrites show excellent separation + / - ( >2 logs) 580V. Whether I
measure APC in FL5 (remove 710LP dichroic, use 670/40BP) or FL6 (710LP plus
670BP), I obtain good APC signal. But no PharRed with the 710LP dichroic /
740EFLP in FL5, and no PharRed signal even if I remove the 710LP dichroic.
BD Pharmingen Technical data sheet recommends a Hammatsu R3896 PMT for
improved red sensitivity. Is the Hammatsu 957-08 deficient in the >740
range?
If not, what's up?
Thanks,
Claude
(See attached file: cantinc.vcf)
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