For determining the pattern of GPCR-arrestin trafficking, HEK293 cells were seeded into collagen-coated 35mm glass bottom Petri dishes (MatTek Corp., Ashland, MA) and co-transfected with 1.3µg of plasmid DNA encoding the receptors of interest and 0.7µg of plasmid encoding green fluorescent protein (GFP)-tagged arrestin319 (link) using FuGene HD. Forty-eight h after transfection, cells were serum derived for 4h, stimulated with a saturating ligand concentration for 8min, fixed with 4% paraformaldehyde in phosphate buffered saline for 30min and washed with 4°C saline. Arrestin distribution was determined by confocal microscopy performed on a Zeiss LSM510 META laser-scanning microscope with 60× objective using 488nm excitation and 505–530nm emission wavelengths. Measurement of AT1AR-Arr3 avidity was performed as previously published27 (link). HEK293 cells stably expressing AT1AR and transfected with Arrestin3-pEYFP were stimulated with AngII (1µM) or analogs (10µM) for 15 min, after which endosomes were bleached and fluorescence recovery was monitored every 30sec over a period of 5 min.
Collagen coated 35mm glass bottom petri dishes
Manufactured by MatTek
Collagen-coated 35mm glass bottom Petri dishes provide a surface for cell culture. The dishes are made of glass with a collagen coating on the bottom, which facilitates cell attachment and growth.
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Lab products found in correlation
3 protocols using collagen coated 35mm glass bottom petri dishes
1
Visualizing GPCR-Arrestin Trafficking Dynamics
2
Visualizing GPCR-Arrestin Trafficking Dynamics
3
Irradiation of Cerebral Endothelial Cells
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HCMEC/D3 (CELLutions Biosystems Inc), an immortalized cell line from cerebral microvascular endothelial cells, were routinely cultured in EBM-2 endothelial basal medium (Lonza) supplemented with 5% fetal bovine serum, 1% Penicillin/Streptomycin, 10 mM HEPES (Life Technologies), and 1 ng/mL human basic fibroblast growth factor (Sigma-Aldrich) at 37˚C in 5% carbon dioxide and passaged at 90% confluence with trypsin-EDTA. All culture vessels were precoated with 100 µg/mL rat tail collagen (In Vitro Technologies).
For irradiation, cells were seeded at 1x10 4 cells/mL onto collagen-coated 35 mm glass bottom petri dishes (MatTek Corporation) containing 1.5 mL EBM-2 medium and allowed to grow for 2 days to achieve 100% confluency. Cells were treated with a single dose of 5, 15 or 25 Gy of ionizing radiation by linear accelerator (LINAC; Elekta Synergy, Crawley, UK) at Macquarie University Hospital (Sydney, Australia), as previously described [11, 13] . Non-irradiated cells treated identically but without radiation (sham) were used as controls. At 1, 3 or 5 days after radiation or sham treatment, the cells were transferred to the parallel-plate flow chamber.
For irradiation, cells were seeded at 1x10 4 cells/mL onto collagen-coated 35 mm glass bottom petri dishes (MatTek Corporation) containing 1.5 mL EBM-2 medium and allowed to grow for 2 days to achieve 100% confluency. Cells were treated with a single dose of 5, 15 or 25 Gy of ionizing radiation by linear accelerator (LINAC; Elekta Synergy, Crawley, UK) at Macquarie University Hospital (Sydney, Australia), as previously described [11, 13] . Non-irradiated cells treated identically but without radiation (sham) were used as controls. At 1, 3 or 5 days after radiation or sham treatment, the cells were transferred to the parallel-plate flow chamber.
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