Quantitative Flow Cytometry

Example from Rebecca Richards-Kortum, Ph.D. and Elizabeth Hsu, University of Texas at Austin, College of Engineering, Division of Biomedical Engineering and Dennis Johnston, Ph.D., SPRD Facility Core 6: Biostatistics and Data Processing.

In the above example the number of anti-Human EGFR sites expressed on the cell surface of Human squamous carcinoma cell line SQCCY1 was quantitated (~ 310,717 EGFR sites/cell) with the DAKO QIFIKIT^®. The above example shows 5 populations of beads that bind calibrated quantities of anti CD5-FITC antibody. This kit is designed for measuring immunofluorescence indirectly.

Quantitative Fluorescence Cytometry (aka Quantitative Flow Cytometry - QFCM) is a dynamic application that enables the Principle Investigator to quickly analyze a large number of cells for multiple antigens and quantitates the number of binding sites per cell. The mean fluorescence intensity (MFI) of a population of cells labeled with a fluorochrome conjugated to a ligand or monoclonal antibody (mAb) is linearly related to the mean number of receptors/cell or mean number of mAb binding sites/cell. Calibrated bead populations that bind a quantifiable amount of antibody (Antibody Binding Capacity - ABC) generate a calibrated standard curve (linear regression) that converts channels of mean fluorescence intensity (MFI) into molecular equivalents of soluble fluorochrome (MESF). The following reference compares the three techniques listed on this web page.

Serke, S., van Lessen, A., and Huhn, D. (1998) Quantitative Fluorescence Flow Cytometry: A Comparison of the Three Techniques for Direct and Indirect Immunofluorescence Cytometry 33:2 p 179-187

Becton Dickinson distributes QuantiBRITE^TM PE beads for estimating the number of antibodies bound per cell (ABC) in cells labeled with Phycoerytherin (PE) conjugated monoclonal antibodies. Each of the four bead populations has a calibrated mean number of bound PE molecules/bead allowing an Investigator to determine the number of bound mAb-PE molecules/cell. With the known amount of fluorochrome to protein (antibody) or F:P ratio one can determine the amount of antibody bound/cell. This kit is designed for measuring immunofluorescence directly.

The Quantum^TM Simply Cellular^® kit distributed by Bangs Laboratories, Inc. provides several different ranges of antibody binding capacity (ABC) 1,000 - 80,000, 5,000 - 150,000, and 10,000 - 200,000 as well as MESF kits. Four bead populations have calibrated amounts of bound Goat anti-Mouse IgG which enable an Investigator to label the beads specifically with any murine monoclonal antibody directed against their species and antigen of interest by direct or indirect labeling as well as with their fluorochrome of choice, i.e. FITC, PE, etc. This kit is designed for measuring immunofluorescence directly.

Additional References

Cytometry (1998) 33:2

Ford, R., Tamayo, A., Martin, B., Niu, K., Claypool, K., Cabanillas, F., and Ambrus, J. (1995) Identification of B-Cell Growth Factors (Interleukin-14; High Molecular Weight-B-Cell Growth Factors) in Effusion Fluids from Patients with Aggressive B-Cell Lymphomas Blood 86:1 p.283-293

Brotherick, ., Lennard, T.W.J., Wilkinson, S.E., Cook, S., Angus, B., and Shenton, B.K. (1994) Flow cytometric method for the measurement of epidermal growth factor receptor and comparison with the radio-ligand assay. Cytometry 16: p. 262-269

Fay, S.P., Posner, R.G., Swann, W.N., and Sklar, L.A. (1991) Real-time analysis of the assembly of ligand, receptor, and G protein by quantitative fluorescence flow cytometry. Biochemistry 30:20 p. 5066-5075

Unckum, F.M., Myers, D.E., Fauci, A.S., Chandan-Langlie, M., and Ambrus, J.L. (1990) Leukemic B-cell Precursors Constitutively Express Functional Receptors for Human Interleukin-1. Blood 74:2 p. 761-776

Uckun, F.M., Fauci, A.S., Chandan-Langlie, M., Myers, D.E., and Ambrus, J.L. (1989) Detection and Characterization of Human High Molecular Weight B Cell Growth Factor Receptors on Leukemic B Cells in Chronic-Lymphocytic Leukemia. J. Clin. Invest. 84:5 p. 1595-1608

Chatelier, R. and Ashcroft, R. (1987) Calibration of flow cytometric fluorescence standards using isoparametric analysis of ligand binding. Cytometry 8:6 p. 632-636

Sklar, L.A., Finney, D.A., Oades, Z., Jesaitis, A., Painter, R., and Cochrane, C. (1984) The dynamics of ligand-receptor interactions. Real-time analyses of association, dissociation, and internalization of an N-formyl peptide and it's receptors on the neutrophil. J. Biol. Chem. 259:9 p. 5661-5669

Bohn, B. (1976) High-sensitivity cytofluormetric quantitation of lectin and hormone binding to surfaces of living cells. Exp. Cell Res. 14: p. 294-306

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