All- Most commercial reagents are sold within 2 to 4-fold of their lowest useful concentration when used according to manufacturer's instructions, so there is some room for dilution but not much. Manufacturer's instructions aside, in most cases it is antibody concentration that matters and NOT amount of antibody per cell. This is because (for the reagents that we have looked at in any case)you are staining in extreme antibody excess. Thus if you want to save antibody the trick is to concentrate the cells. If you reduce the staining volume by a factor of 10 you can usually reduce the amount of antibody commensurately. We usually stain "dry cell pellets" in microtiter wells. After spinning and "flicking" the average well volume is 10 mcl (whether we have added 300,000 or 5 million cells per well). Thus in many cases we can get by with 1 or 2 mcl of antibody (yes, even for 5 million cells). As always, there is no getting around doing a titration curve. If you don't have a "plate swinger" you can still stain dry pellets in 12x75 tubes, but the staining volume is a little larger. Albert D. Donnenberg, Ph.D. -----Original Message----- From: cheryla.smith@utoronto.ca [mailto:cheryla.smith@utoronto.ca] Sent: Monday, February 21, 2000 12:36 PM To: cyto-inbox Subject: dilution of antibody Hello out there, One of the users of our facility has a concern regarding the advisability of diluting a particular antibody. Since these reagents are quite expensive she doesn't want to go ahead assuming that all will be well. Please respond to her following question directly at: sandra.aswald@utoronto.ca Thanks I am planning to use the antibody: anti-human IL-12 (p40/p70) mouse IgG FITC; cat.# 18994A from Pharmingen. It should be used at aprox. 0.5ug per 10 million cells, and comes as 0.1mg in a total volume of 200ul. I was wondering a) use only 1ul undiluted AB per 10 million cells b) dilute 1:10 and use 10ul Question: .Since I will be doing intracellular FACS with permeabilized cells, is it O.K. to dilute the antibody? .If I can dilute it, is there an alternative to using the Pharmingen Perm/Wash (TM) buffer? This is the only thing that Pharmingen suggests diluting their anti-cytokine AB's with. Thanks for your help Sandra
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