Visualizing Receptor Dynamics in Live Cells

Prior live-cell imaging, 24 h post-transfection, Cos-7 or HUVEC cells grown on 25 mm #1.5H glass coverslips (Thorlabs, CG15XH1) were incubated for at least 30 min in serum-free FluoroBrite DMEM media (Gibco, A1896701) supplemented with 5 μM biliverdin (co-factor for iRFP713; Cayman Chemical, 1925710). Time-lapse TIRF imaging was performed following stimulation with hEGF (100 ng/ml^{21 (link)}; Millipore Sigma, E9644-.2MG) for Cos-7 cells, or recombinant hVEGF 165 (50 ng/ml^{60 (link)}; Fischer Scientific, 293VE010CF) for HUVECs. Each cell was imaged simultaneously with TIRF 488 and TIRF 647, for 5 min with 200 ms exposure time and 100 ms delay between frames. Three to four cells were imaged per ligand stimulation.

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Nawara T.J., Williams YD I.I., Rao T.C., Hu Y., Sztul E., Salaita K, & Mattheyses A.L. (2022). Imaging vesicle formation dynamics supports the flexible model of clathrin-mediated endocytosis. Nature Communications, 13, 1732.

Publication 2022

CG15XH1 FluoroBrite DMEM media biliverdin

Biliverdin Cayman Cells Cos 7 cells Frames Huvec cells Ligand Serum free media Transfection

Corresponding Organization :

Other organizations : University of Alabama at Birmingham, Emory University

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Variable analysis

independent variables

HEGF (100 ng/ml) for Cos-7 cells
Recombinant hVEGF 165 (50 ng/ml) for HUVECs

dependent variables

Time-lapse TIRF imaging

control variables

Cos-7 or HUVEC cells grown on 25 mm #1.5H glass coverslips (Thorlabs, CG15XH1)
Incubation in serum-free FluoroBrite DMEM media (Gibco, A1896701) supplemented with 5 μM biliverdin (co-factor for iRFP713; Cayman Chemical, 1925710) for at least 30 min prior to imaging
Simultaneous imaging with TIRF 488 and TIRF 647, for 5 min with 200 ms exposure time and 100 ms delay between frames
Imaging of 3 to 4 cells per ligand stimulation

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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