Live-cell imaging of ER-localized proteins

CHO cells were transfected with mCherry-ER and YFP-AT or YFP-AAT constructs in 35 mm live-cell imaging dishes (MatTek) and, 24 hours later, imaged in tissue culture medium supplemented with 25 mM HEPES, pH 7.0 to 7.6. Live-cell imaging was performed as reported previously (33 (link), 62 (link)). Confocal micrographs were acquired using a Zeiss LSM780 laser scanning confocal microscope (Zeiss) using a ×63 1.4NA oil immersion objective. YFP- and mCherry-tagged proteins were excited at 514 nm and 561 nm, respectively, and images were captured with 1,028 × 1,028 pixel frame size.

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Bravo-Pérez C., Toderici M., Chambers J.E., Martínez-Menárguez J.A., Garrido-Rodriguez P., Pérez-Sanchez H., de la Morena-Barrio B., Padilla J., Miñano A., Cifuentes-Riquelme R., Vicente V., Lozano M.L., Marciniak S.J., de la Morena-Barrio M.E, & Corral J. (2022). Full-length antithrombin frameshift variant with aberrant C-terminus causes endoplasmic reticulum retention with a dominant-negative effect. JCI Insight, 7(19), e161430.

Publication 2022

35 mm live-cell imaging dishes Zeiss LSM780 laser scanning confocal microscope

Cell Cho cells Culture medium Dishes Frame Hepes Immersion Laser scanning microscope Proteins Tissue

Corresponding Organization : Universidad de Murcia

Other organizations : University of Cambridge, Universidad Católica San Antonio de Murcia

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Variable analysis

independent variables

Transfection of CHO cells with mCherry-ER and YFP-AT or YFP-AAT constructs

dependent variables

Live-cell imaging of transfected CHO cells

control variables

Imaging was performed in tissue culture medium supplemented with 25 mM HEPES, pH 7.0 to 7.6
Confocal micrographs were acquired using a Zeiss LSM780 laser scanning confocal microscope with a ×63 1.4NA oil immersion objective
YFP- and mCherry-tagged proteins were excited at 514 nm and 561 nm, respectively, and images were captured with 1,028 × 1,028 pixel frame size

controls

No positive or negative controls were explicitly mentioned in the provided information.

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