23 May 1997 Time-dynamic imaging of individual cell ligand binding kinetics
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Proceedings Volume 2983, Functional Imaging and Optical Manipulation of Living Cells; (1997) https://doi.org/10.1117/12.274332
Event: BiOS '97, Part of Photonics West, 1997, San Jose, CA, United States
Ligand-binding assays are commonly applied to large numbers of cells in culture; the binding parameters derived from such assays reflect the ensemble average behavior of many cells. Equilibrium binding assays of epidermal growth factor (EGF) binding to the EGF receptor (EGFR) indicate that the EGFR exhibits two affinity states for EGF, one low affinity with Kd about 10 nM and one high affinity with Kd < 1 nM. Bulk binding studies cannot determined if such multiple ligand binding classes are due to cell population heterogeneity or are due to heterogeneity at the individual cell level. Here is described a technique based on single cell imaging of fluorescein-EGF (f-EGF) binding to individual human epidermoid carcinoma A431 cells that demonstrates that both classes of EGFR are found on all A431 cells, that the time course of f-EGF binding to individual cells shows two kinetic on-rates and two off-rates, that cell-to-cell heterogeneity of EGF binding is significant and that ligand binding kinetics vary across an individual cell. Contributions of cell autofluorescence photobleaching and f- EGF photobleaching in the measurement of fluorescent ligand binding are shown to be significant.
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David Gross, David Gross, Johnson Chung, Johnson Chung, } "Time-dynamic imaging of individual cell ligand binding kinetics", Proc. SPIE 2983, Functional Imaging and Optical Manipulation of Living Cells, (23 May 1997); doi: 10.1117/12.274332; https://doi.org/10.1117/12.274332

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