What happens if a suboptimal concentration of a primary antibody is used?
The ideal concentration of primary antibody leads to bright labeling of target cells combined with the least possible non-specific staining of non target cells, measurable by flow cytometry. • Too high concentrations of primary antibody may lead to unspecific labeling of non-target cells. In such a case, target cells as well as non-target cells will be magnetically labeled during the incubation with MACS® MicroBeads for indirect magnetic labeling. This will reduce purity of the selected positive fraction. In case of a depletion strategy, it may lead to a lower recovery of the unlabeled target cells. In both cases, a certain amount of the undesired unspecifically magnetically labeled cells will be retained on the column. • Too low concentration of primary antibody can lead to an insufficient magnetic labeling of target cells, resulting in low recovery of the positive fraction (positive selection) or in reduced purity of the negative fraction (depletion).
