Hi - What follows is a conversion between the integrated PMT holders that Cytomation uses and the Hamamatsu tube types. Description Hamamatsu Tube Type PMT & Mounting, H957-06 931B PMT & Mounting, H957-08 R928 PMT & Mounting, H957-12 R1477 PMT & Mounting, H957-15 R3896 You have an R928 type tube. That is significantly less sensitive than the R3896 type tubes, not only in the longer red wavelengths, but in all measurement channels from FITC on out. We had a 1477 type on the Cy7APC (PharRed) channel here and we could not see separation between the positive and negative peaks of CD8 stained lymphs. Upgrading the PMT to a 3896 tube fixed that (although that measurement channel is not as good as some others). We also switched the PMTs on all the channels (except side scatter) to the 3896 type, and saw an improvement on each. On the Vantage here we use a HeNe at 35 mW and a filter set similar to yours. To check for staining problems you could first bulk stain cells with a biotinilated or unconjugated antibody, alloquat into separate tubes and follow by second steps with different conjugations. >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 > > > > > > > > >Content-Type: text/x-vcard; charset=us-ascii; > name="cantinc.vcf" >Content-Transfer-Encoding: 7bit >Content-Description: Card for Claude Cantin >Content-Disposition: attachment; > filename="cantinc.vcf" > >Attachment converted: Asimov:cantinc.vcf (TEXT/ttxt) (0001732F) -- Marty Gladstone Institutes Flow Core mbigos@gladstone.ucsf.edu 695-3832
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:57:40 EST