Optimal Loading of Scraped HeLa Cells with Monoclonal Antibodies to the Proliferation-associated M_r 120,000 Nucleolar Antigen¹

Our laboratory has reported (J. W. Freeman et al., Cancer Res., 48: 1244–1251, 1988) a proliferation-associated M_r 120,000 nucleolar antigen (p120), that was found in human tumors but was not detectable in most normal resting tissues and in benign tumors. To study further the function and the localization of this protein, we have investigated various methods of microinjecting p120 monoclonal antibodies into cells. For comparison, we have used a monoclonal antibody to protein C23, a nucleolar protein found in high levels in most cells.

To determine optimal conditions for loading of antibodies to nucleolar antigens into mechanically disrupted HeLa cells, we studied the effects of ionic strengths of loading buffers, various antibody concentrations, and optimal time for loading and antibody localization. With ascites fluids in isotonic buffer containing antibodies to nucleolar proteins p120 and C23, a maximum number of cells, 86 and 84%, respectively, were loaded following a 20-min incubation. Hypotonic buffers decreased the percentage of cells loaded (22%); hypertonic buffers reduced cell viability.

The optimal concentration of purified antibody yielding a maximum number of loaded cells (81%) was 2.5 mg/ml. Higher concentrations of antibody resulted in residual cytoplasmic staining without increasing the percentage of loaded cells. With antibody concentrations less than 2.5 mg/ml, a linear decrease was noted in the percentage of cells loaded with a decrease in intensity of fluorescence.

Following antibody loading, nucleolar fluorescence was observed by 12 h and the intensity increased at 24 h. Localization of the p120 antibody was followed through mitosis where it was perichromosomal and equally divided between the chromosomes at metaphase. A decrease of nucleolar immunofluorescence intensity and percentage of cells labeled were observed in successive cell generations.