The In-Cell ELISA principle is similar when using either IR or colormetric detection methods. After cells have been grown to approximately 80% confluency in a 96- or 384-well plate, a drug or other treatment is applied to stimulate a cellular response. After treatment the cells are fixed and permeabilized in the wells, effectively "freezing" the cells in context with no further sample prep perturbations. Primary antibodies are then added which bind to their intended targets within the mitochondria or other subcellular compartment. After incubation, the unbound primary antibodies are washed away and secondary antibodies are added. These secondaries are conjugated to either IRDyes® or to an enzyme label (HRP or AP) for the colorimetric versions of the assays. Unbound secondaries are washed away, reaction buffer is added for the colorimetric assays, and the signal is read on a suitable instrument for the kit type.
