Immunofluorescent quantification of ribonucleotide reductase M1 subunit and correlation with DNA content by flow cytometry.
The cytoplasmic enzyme ribonucleotide reductase is essential for DNA synthesis, and its activity is strongly correlated with cellular proliferation. This paper describes a flow cytometric technique for the simultaneous measurement of DNA content and the M1 subunit of ribonucleotide reductase. Data are presented for cycling cultured human leukemic lymphoblasts in which M1 is constitutively expressed, and peripheral blood lymphocytes in which it is only detectable with certainty after mitogen stimulation. The choice of fixation procedure strongly influenced the amount of M1 subunit detected. Paraformaldehyde (PF) at concentrations of 2% (w/v in PBS) or greater provided optimal results. Fixation at 37 degrees C was significantly more effective in preserving M1 than fixation at room temperature or 4 degrees C. These variables are shown to have affected cytoplasmic retention during postfixation processing. Their relevance to the flow cytometric measurement of other intracellular components by this procedure are discussed.
Mann
G J
GJ
Ludwig Institute for Cancer Research (Sydney Branch), University of Sydney, New South Wales, Australia.
Dyne
M
M
Musgrove
E A
EA
D002453:Cell Cycle; D003593:Cytoplasm / Q000201:enzymology; D004273:DNA, Neoplasm / Q000032:analysis*; D005404:Fixatives; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D005557:Formaldehyde; D006801:Humans; D007945:Leukemia, Lymphoid / Q000201:enzymology / Q000378:metabolism; D008214:Lymphocytes / Q000032:analysis / Q000201:enzymology; D008854:Microscopy, Electron; D011108:Polymers; D011419:Propidium; D012264:Ribonucleotide Reductases / Q000032:analysis*
D004273:DNA, Neoplasm; D005404:Fixatives; D005456:Fluorescent Dyes; D011108:Polymers; D005557:Formaldehyde; D011419:Propidium; D012264:Ribonucleotide Reductases; C003043:paraform