Flow cytometric measurement of RNA synthesis using bromouridine labelling and bromodeoxyuridine antibodies
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Flow cytometric measurement of RNA synthesis using bromouridine labelling and bromodeoxyuridine antibodies. / Jensen, P O; Larsen, J; Christiansen, J; Larsen, J K.
In: Cytometry, Vol. 14, No. 4, 1993, p. 455-8.Research output: Contribution to journal › Journal article › peer-review
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TY - JOUR
T1 - Flow cytometric measurement of RNA synthesis using bromouridine labelling and bromodeoxyuridine antibodies
AU - Jensen, P O
AU - Larsen, J
AU - Christiansen, J
AU - Larsen, J K
PY - 1993
Y1 - 1993
N2 - 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.
AB - 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.
KW - Antibodies, Monoclonal
KW - Bromodeoxyuridine
KW - Flow Cytometry
KW - Fluorescein-5-isothiocyanate
KW - Fluorescent Antibody Technique
KW - Humans
KW - Leukemia, Promyelocytic, Acute
KW - RNA
KW - RNA, Neoplasm
KW - Ribonucleases
KW - Tumor Cells, Cultured
KW - Uridine
KW - Comparative Study
KW - Journal Article
KW - Research Support, Non-U.S. Gov't
U2 - 10.1002/cyto.990140416
DO - 10.1002/cyto.990140416
M3 - Journal article
C2 - 7685681
VL - 14
SP - 455
EP - 458
JO - Cytometry
JF - Cytometry
SN - 0196-4763
IS - 4
ER -
ID: 181874021