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Staining Volume

Many staining reagents, particularly monoclonal antibodies, are expensive. Any protocol for staining cells should have expense as one of the criteria for experimental design. Although the number of cells for an experiment is constrained by both experimental and flow cytometer design and the concentration of stain used is constrained by a desire to achieve near saturation (see above), the staining volume is under user control. The smaller the staining volume, the less stain will be required to achieve saturating concentration. Although many companies selling staining reagents suggest staining in 100 ml volume, it usually works to reduce this to 50 ml or even less. This reduces the requirement for stain by 2- or more-fold. The only considerations here are that you don't go to such a small volume that pipetting becomes inaccurate, nor that the amount of stain becomes limiting (that is, that there isn't enough stain to go around to all the receptors on all the cells present). With 50 ml of stain at a saturating concentration, 10^6 cells with antigens within the usual range of density will not use up a significant fraction of most antibodies. Check this out by using 2-fold more and 2-fold fewer cells than you are ever likely to have and convince yourself that the fluorescence intensity of the cells in all cases is the same.