• Flow cytometry;
  • population identification;
  • enzyme kinetics;
  • membrane enzymes


Two flow cytometers, an Ortho Cytofluorograf model 4800A and a “custom” built dual laser instrument, have been interfaced with a PDP 11/40 computer to assay the rate of accumulation of fluorescent products in populations of single cells due to enzymatic hydrolysis of fluorogenic substrates. Esterase activity assayed with fluorescein diacetate exhibited abnormal Michaelis-Menten kinetic behavior in EMT6 cells. This was in marked contrast to normal kinetics obtained with homogenates assayed with a conventional spectrofluorimeter. Moreover, the homogenates were 4 5x less efficient at converting substrate than were intact cells. Fluorescein diacetate is lipophilic and the induced fluorescence was evenly distributed throughout intact cells. In contrast a second substrate studied, 3-O-methyl fluorescein phosphate, is lipophobic and highly polar and the induced fluorescence was located at the cell surface. Results obtained with this substrate suggested that membrane, but not intracellular phosphatases were being assayed. Although the reaction product appeared to be lost rapidly from the site of hydrolysis it was possible to extract Km and Vmax for the dephosphorylation which followed normal kinetics. The dual laser flow cytometer was built to study 4-methylum-belliferone based substrates (UV excitation) simultaneously with those based on fluorescein. An assay using fluorescein diacetate and 4-methylumbelliferyl acetate simultaneously was able to discriminate between a mouse mammary tumour (EMT6) and a human colonic adenocarcinoma cell line (HT 29). Furthermore, incubation of both cell lines for up to 6 hr with these substrates maintained viability at about 90%.