Immunolocalization of Rh and RhAG in K562 transfectants.

TPA-treated and untreated cells were incubated with mMAb LA18.18 and hMAb LOR-15C9. MAbs bound to RhAG and RhD polypeptides were probed with Alexa-Fluor 488–conjugated anti-mouse IgG (green fluorescence) and Alexa-Fluor 568–conjugated anti-human IgG (red fluorescence), respectively. Fluorescence images were visualized using a confocal laser microscope. Coexpression of Rh and RhAG resulted in yellow fluorescence on merged images. Because of the small size and high nucleus/cytoplasmic ratio of K562 cells, discriminative fluorescence staining patterns can be interpreted with confidence only for expression or nonexpression of the relevant antigens at the cell membrane.