Flow cytometric measurement of RNA synthesis using bromouridine labelling and bromodeoxyuridine antibodies

Nuclear RNA synthesis can be analysed by flow cytometry of cells labelled with 5-bromouridine (BrUrd) and stained with anti-bromodeoxyuridine (BrdUrd) antibody and FITC-conjugated secondary antibody. A panel of 5 different commercially available anti-BrdUrd antibodies was tested on cells of a HL-60 human leukemia cell line, stained as a methanol-fixed nuclear suspension. The BrUrd-induced fluorescence signals were highest with the antibody ABDM (Partec), moderate but reproducible with B-44 (Becton Dickinson), variable or low with BR-3 and IU-4 (Caltag), and not detectable with Bu20a (DAKO). Treatment of BrUrd-labelled nuclei with ribonuclease before staining with antibodies indicated that ABDM and B-44 antibodies specifically recognized BrUrd-substituted RNA, whereas BR-3 and IU-4 antibodies also bound to BrUrd-unlabelled RNA. Combined analysis of BrUrd and DNA contents demonstrated the variation of RNA synthesis during the cell cycle. The BrUrd incorporation was high in the S and G2 phase, variable in G1, and negligible in mitosis. Similar results were obtained using other cell types.