From: Mario Roederer (Roederer@Darwin.Stanford.EDU)
Date: Wed Feb 25 1998 - 18:28:19 EST
> Can anyone shed some light on how they handle specimen > that has very low cell count e.g. <2500/mm3, CSF, FNA > etc. Do you dilute antibody concentration or cut back > amount of antibody to accomodate the same ratio of > cell:antibody? No! Do NOT keep the same ratio of antibody to cells in staining protocols involving different numbers of cells! Rather, keep the antibody concentration the same (i.e., use the same amount of antibody). At cell numbers under about 20 Million per stain, the primary parameter affecting final fluorescence is the concentration of the antibody--and is essentially independent of antigen concentration. (This assumes that you are using a "typical" antibody, at about 1 ug per test). At 1M cells or less, the antibody is in very high excess over the antigen; the stain time is driven primarily by the on-rate. Thus, using less antibody will result in less fluorescence even with fewer cells present. Likewise, there is no need to increase the antibody unless you are using well over 20M cells per test--and even then, you only need to increase it a few-fold. These issues are covered in detail in a chapter by Kantor & Roederer in the most recent Handbook of Experimental Immunology. It is always best to titer your reagents under the conditions that you intend to use them. mr
This archive was generated by hypermail 2.1.6 : Thu Jan 01 2004 - 17:35:13 EST
