Quantitative analysis of a nuclear antigen in interphase and mitotic cells

Abstract

The quantification of an interchromatin-as-sociated antigen, designated p105, during cellular passage through mitosis is described. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated a qualitative increase in p105 within the mitotic cytoplasm. Multiparameter flow cytometric analysis was performed on fixed cells sequentially stained with anti-p105 immunofluorescence and/or propidium iodide. This analysis demonstrated approximately a tenfold increase in intracellular p105 content as a function of progression from the G₂ to the M phase. This increase was corroborated by the quantitative immunoblot analysis of colchicine-treated cell cultures and of cells sorted on the basis of anti-p105 immunofluorescence. The data reveal that the increased levels of anti-p105 immunofluorescence in conjuction with flow cytometry may be used effectively to quantitate mitotic index and isolate mitotic cells. The function and modulation of p105 throughout the cell cycle is discussed.