At 09:49 7/6/99 -0400, G Robin Barclay wrote: > >Dear colleagues >Can anyone recommend a method that works for them for quenching FITC-tagged >agent when bound to the external cell surface but not when the agent is >internalised by the cell. Trypan blue and crystal violet come to mind - >does anyone have this up and running - can you give method/recipes? I >assume the quenching agent has to be present in the final cell suspension >to be run on the cytometer - is it just a matter of resuspending the washed >FITC-labelled cells in a quencher solution (in PBS?) and running them >immediately - or are there finite times/temperatures involved. Is it true >that only Merck trypan blue works? Has anyone compared trypan blue to >crystal violet - and which came out best? Are there other quenchers which >may be better? Do any of these quenchers significantly contaminate the >cytometer tubing - how do you clean after running? Robin: In addition to trypan blue, two thoughts come to mind... 1) I think it is BMB (or now Roche) and /or Sigma that sells a monoclonal antibody to FITC and that fluorescence is markedly diminished when the MAb is bound. 2) I've heard that EtBr can also quench FITC, by energy transfer, as well but that's OK as long as you don't need FL2. But then one might want to use FL2 to monitor quenching. I can't speak to how or if EtBr would "coat the lines" of a cytometer. Hope this helps, David ============================= David L. Haviland, Ph.D. Asst. Prof. Immunology University of Texas - Houston, H.S.C. Institute of Molecular Medicine 2121 W. Holcombe Blvd. Houston, TX 77030 Internet:"dhavilan@imm2.imm.uth.tmc.edu" Voice: 713.500.2413 FAX: 713.500.2424 ------------------------------------------------------ The hardness of butter is directly proportional to the softness of the bread. =============================
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:53:41 EST