The rate of disappearance and reexpression of surface immunoglobulin (Ig) and Ia-like antigen (Ia) on blood lymphocytes obtained from 10 healthy individuals during 4 days of in vitro incubation was investigated. Membrane antigens were "prelabeled" with fluoresceinated antibodies, cells were cultured in duplicate and examined daily. The proportion of prelabeled cells diminished almost completely by Day 3. Studies employing radiolabeled instead of fluoresceinated antibodies revealed a progressive increase in radioactivity in the culture medium, suggesting that the decrease in antibody-prelabeled cells was due primarily to antigen shedding. To confirm this idea, antigen chemically conjugated to polyacrylamide beads was reacted with radiolabeled antibodies and added to cell cultures. A significant release of radioactivity into culture medium was not observed, verifying that shedding of the cell membrane antigen, not degradation of antibody or dissociation of antigen-antibody complexes, was the mechanism involved in the decrease of the prelabeled cells. In duplicate cultures, cells were restained daily to determine the sum of remaining prelabeled cells and the cells which newly expressed antigen during culture ("daily labeled" cells). A similar proportion of daily labeled la-bearing lymphocytes was found throughout the culture, whereas the proportion of daily labeled Ig-bearing cells decreased significantly by Day 3. The decrease was unrelated to cell viability or antibody prelabeling. The result suggests that the in vitro culture environment altered reexpression of Ig, but not Ia. This antibody prelabeling and culture procedure provides a dependable and physiologic method of removing membrane molecules and of investigating factors which influence endogenous surface antigen expression.
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