Friends: Thankyou very much for the suggestions and input. As many of you were interested in the mAb titration subject I have included all the input I received below. Since I had about 30 mAbs to titrate I was very happy to see Mario's reponse that 4-color titration was doable. Anyway I tried this out combining it with the other suggestions to calculate signal to noise ratios using the arithmetic mean values on individual histograms at each mAb dilution. Each panel of 4 mAbs were mixed at the same conc., 6 dilutions in all. I used unlabelled cells as background, since my mouse PBLs and splenocytes give high backgrounds with isotype controls (in spite of Fc block). Acquistion was done keeping the Detector/Amplification values constant for a panel, but compensating at each subsequent dilution. The Detector/PMT values were set to get the best resolution at maximum mAb concentration (1ug/million cells). Most of the mAbs showed an increse in signal/noise ratio with conc. then a plateau over 2-3 concs. and sometimes, another increase to the highest conc. I selected as optimal the lowest conc. required for the plateau value. The logic being that signal could be increased by changing detector settings and compensation would be less of a nightmare. I will know if these selected concs. work in the 4-color mix in a day or two. All comments and criticisms are welcome! Yasmin a)"Dennis Broud 301-594-5879 FAX 301-594-3037" <BROUDD@cder.fda.gov> titrate them individually first. I generally use 10 ul of the AB dilutions to 100 ul of whole blood, if I'm lysing. Or if I have prepared cells, 10 ul of ab to 100ul of cells (about 1,000,000 cells). I generally start at 1:20 and do two fold dilutions up to 1:640 as a starting off place for commercial antibodies, unless the manufacturer has already specified a dilution. Once I've titered each antibody this way, I've found that I can generally add all 3 or 4 antibodies together into a pool. That is use 30 or 40 ul of AB mixture to 100ul of whole blood or to 1 million cells. I let all my staining go for at least 30 minutes before I lyse wash, etc. If cells have been stimulated in any way (especially mouse cells) I block with 40 ul of 1:40 dilution of FC block (Pharmingen)to the cells for 15 minutes at room temperature and then add the antibody cocktail. Occasionally you'll get a wierd antibody that will act differently in a cocktail than it does by itself, then you have to do all the combinations to figure out what's going on, but fortunately that's not very often. b) "Dennis J. Young" <djyoung@ucsd.edu> I'd titrate individually, with the assumption that stearic hindrance is minimal. Start at 200 - 300 micrograms per ml of Ab and use 6 serial 1:3 dilutions plus an unlabeled control. So you'd need to run only 25 tubes (6*4 + 1) Measure signal to noise, not the "brightest" positives. Then I would verify the 4-Ab cocktail. See Chapter 4.1 in Current Protocols in Cytometry (C.C. & S.J. Stewart) c) Mario Roederer <Roederer@DrMR.com> We often combine and titre as you suggest--as long as the antibodies don't cross block (i.e., don't titre FITC CD8 with PE CD8, etc.), and as long as you set proper compensation. Then it really doesn't matter, and you can titre all four effectively on the same cells. d) Maciej Simm <simmmmer@yahoo.com> I've only titrated my AB's 1 at a time and mixed them at the lowest titration that gave me the same signal as stock.
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