From: Mario Roederer (ROEDERER@Darwin.Stanford.EDU)
Date: Tue Nov 08 1994 - 16:21:53 EST
We also typically do 2-fold dilutions. It is important to include a population of cells that will not stain with the mAb in order to assess background "stickiness" at the same time as true staining. BTW, we find that for most antibodies, the cell number being stained is relatively unimportant (i.e., the amount of antibody is in vast excess over antigen). However, this may not be true of all antibodies. You should use consistent cell numbers (1 million per test is generally used), and do a test to make sure that a sample with extra cells (3-5 million) has the same staining profile. We also fit standard Michaelis binding curves to our data; you can use Kaleidagraph or JMP [=SAS for Macs] (or for those with Consort-40 or -VAX, you can use COTFIT) to do nonlinear least squares fitting of any function. For those of you who will test manufacturer's reagents, you will find that most of the phycobilli-conjugates (PE, Cy5-PE) are packaged well below saturating concentrations. Most FITC and Biotin reagents are packaged at saturating concentrations. For some conjugates, this is done because saturating concentrations would make cells off-scale in fluorescence (especially for Cy5-PE). Our approach is to "cut" these reagents with unconjugated (i.e., cold competitor) such that the final concentration of mAb is saturating and the final fluorescence is on-scale. It is important to understand the consequences of being below saturating concentrations of these reagents: the final fluorescence that will be obtained will be proportional to (1) the amount of mAb added, and (2) the time of incubation with the antibody. Again, the cell number usually isn't a factor, because even at subsaturating concentrations that mAb can still be in vast excess over antigen. If you are doing antigen density measurements, it becomes essential to have saturating concentrations of reagent! mr
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