Why do I need to count my cells?

Binding of a (ligand) fluorochrome to DNA essentially follows the law of mass action. One can calculate approximate concentrations of the ligand and relate to the cell number to find out the range when there may be a significant decrease in free ligand concentration that may affect DNA stainablility.

There are 3x10E9 DNA base pairs per cell (diploid cell in G1). Most intercalators reacting with free (naked) DNA at saturation bind every second base pair. Thus, potentially, in a single cell there is 15x10E8 binding sites. However, because within the cell some nucleosomal DNA is inaccessible to the intercalators (Cytometry, 5:355-363,1984) only a fraction of the potential binding sites (10-70%, depending on the ligand) actually can bind the ligand. Thus, in a single cell there is maximally between 1.5 to 10 x 10E8 sites that bind these ligands.

Assuming average MW of most DNA fluorochromes to be about 300, one can estimate (from Avogadro number) that at a concentration 100 uM (30 ug/ml) in one ml there are 6x10E16 molecules of the ligand. One million cells has between 1.5 to 10x10E14 binding sites. Thus, there is an nearly 100 times excess of the ligand per binding site when one million cells are stained in 1 ml volume at 100 uM dye concentration.

One would expect that under these conditions a change in cell number from 1 to 2 million (which will change a concentration of the free ligand by 1%) should not be reflected by greater than 1% change in stainability of DNA. However, at lower dye concentration (e.g. below 20uM) or when cell number is more drastically changed (e.g. from 1 to 5 million) the change in DNA stainability, clearly, is noticeable. Needless to say, if cells have higher DNA content, (tetraploid, arrested in G2/M) the equilibrium is shifted towards lesser concentration of free dye, which also may lead to lowed DNA stainability.