Why Is Antibody Titration Important?
To optimize staining in flow cytometry antibody titration is recommended. Whilst antibodies will bind to high affinity targets on a cell, if they are present in excess they will also bind to low affinity targets.
This results in an increase in background fluorescence and consequently a reduction in your ability to resolve populations, especially if there are subtle differences. Furthermore if the antibody concentration is too high, it may result in a false negative prozone effect.
It is therefore important to use the antibodies at the right concentration. Although antibodies are sold with a recommended dilution and it is a good starting point, it may not be optimal for your cell type or protocol, therefore titration is an essential step to optimize your staining.
How to Titrate Your Antibodies
A titration experiment starts by selecting a fixed incubation time, cell type and experimental conditions. The last two should preferably match your final experiment. The cells are then stained in a series of dilutions of the antibody. It is good practice to add a viability dye even when titrating antibodies as dead cells can bind antibodies non-specifically, making your results hard to interpret. To determine the best antibody concentration, the stain index can be used as a guide (formula in Figure 1).
To obtain a stain index you must have a positive and a negative population (Figure 1). If all of your sample population is positive, for instance CD45 on human peripheral blood, then it is necessary to spike the population with some unstained cells. As with all flow cytometry experiments, don’t forget to include a single cell gate in your analysis to exclude doublets.