Titration of fluorochrome-conjugated antibodies for labeling cell surface markers on live cells

Nonspecific antibody binding is best eliminated by optimizing the amount and concentration of the antibody. An antibody titration assay should be applied to determine the antibody amount and concentration resulting in the highest signal of the positive population and the lowest signal of the negative population. While conventional antibody titration protocols focus on the concentration of the antibody, this protocol for antibody titration considers the antibody concentration, as well as the antibody amount. Thus, it is designed to find the optimal antibody concentration for labeling antigens expressed on the surface membrane of live cells, while nonspecific antibody binding is kept to a minimum.