You might be interested in a version of this experiment that we published back in 89: Edwards, B.S., Shopp, G.M. Efficient use of monoclonal antibodies for immunofluorescence. Cytometry 10:94-97, 1989. - Bruce >>> "Donnenberg, Albert" <donnenbergad@MSX.UPMC.EDU> 02/28/00 09:37AM >>> Joost, I think that you should just do the experiment (we have). Add graded numbers of cells to microtiter wells (100K to 10M per well). Stain your cells as dry pellets (obtained by spinning the plates at 700 RPM [~80xg]and then flicking and blotting) using graded amounts of antibody (1 - 10 microliters). You will be amazed. Albert -----Original Message----- From: Joost Schuitemaker [mailto:J.H.Schuitemaker@amc.uva.nl] Sent: Thursday, February 24, 2000 3:35 AM To: cyto-inbox Subject: RE: dilution of antibody 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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