Considering the concentration of 1.5ug antibody per ml to reach reasonable staining saturation within less than 30 minutes as a ballpark figure, this is equivalent to 10nM of antibody. This means that in 1ml there are 10^18 molecules hopping around, or 10^12 antibodies hitting each cell if you have a million cells in there. One can calculate further how many antibodies are within a certain diffusion distance of each cell to get even more precise, but the number of molecular involved might already help to illustrate the funny effects of on and off rates, depletion and mass transport limitations. They are also an important factor to consider when working with particle based assays. In general lower concentrations mean longer incubation times, so do lower temperatures as they reduce the 'hit-rates'. If cells are unfixed that in turn can have effects on cell stimulation, change binding site densities.... so it is not at all easy to decide what to do. Regards Gerhard -----Original Message----- From: Mario Roederer [SMTP:Roederer@drmr.com] Sent: Friday, February 25, 2000 10:09 PM To: Cytometry Mailing List Subject: RE: dilution of antibody Joost: >Or am I mistaken? You are mistaken. Aaron Kantor and I went into this topic in some depth in our chapter in Handbook of Experimental Immunology (5th ed) "FACS Analysis of Leukocytes". I recommend that you look into it for a thorough description. Albert was correct in his analysis--for nearly all antibodies, the number of cells is irrelevant (until you get to numbers of 10's of millions that bind the antibody); the concentration of the antibody, however, is paramount. (Incidentally, so is time and temperature--make sure that you titrate your antibodies at the same temperature and for the same amount of time as your experiments). I think the reason for the origin of this error is that for a long time, manufacturers gave their effective titres as "ug per million cells" rather than "ug per ml". mr (PS: cell number can become relevant for very high affinity antibodies. This is because with high affinity antibodies, the amount of antibody supplied can be much lower, thus, in much lower excess over antigen. However, the vast majority of antibodies have an affinity low enough such that a significant excess of antigen (in typically staining experiments) is required to achieve near-saturation.). >Dear Albert, > >Sorry, but I don't agree with you. When you should look at a marker 80% of >your cells is positive for. And you determine the amount of antibody >(= your >concentration) for 300.000 cells. It is not possible to get even a >peak far >from your negative peak when you stain 5 million cells. This means >that you >throw away at least 5 times the amount of antibody when you stain 300.000 >cells. Because you determined (titrated) your antibody for 5 million >cells. >And the definition of concentration is the amount of Ab in a certain >volume. >So when you got 100.000 cells and need 0.01 µg of Ab to stain them you can >add this in 50 µl or in 5 ml. The only difference is the timeperiod >it will >take to get your Ab on the cells. Or am I mistaken? > >Best regards, > >Joost Schuitemaker
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