ACKR3-APEX2 β-arrestin ½ KO Cells were plated in rat-tail-collagen-coated 35 mm glass bottomed dishes (MatTek Corporation, Ashland, MA). Cells were imaged with a Zeiss CSU-X1 spinning disk confocal microscope using the corresponding lasers to excite GFP (480 nm). Confocal images were arranged and analyzed using ImageJ (NIH, Bethesda, MD).
Rat tail collagen coated 35 mm glass bottomed dishes
Manufactured by MatTek
The Rat-tail-collagen-coated 35 mm glass bottomed dishes are a type of cell culture dish with a glass bottom coated with rat tail collagen. This coating is intended to facilitate cell attachment and growth.
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4 protocols using rat tail collagen coated 35 mm glass bottomed dishes
1
ACKR3-APEX2 β-arrestin Localization
2
CXCR3-Mediated β-Arrestin-2 Recruitment
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HEK293 cells plated in rat-tail-collagen-coated 35 mm glass bottomed dishes (MatTek Corporation, Ashland, MA) were transiently transfected using FuGENE 6 with either wild-type CXCR3-GFP or the indicated CXCR3-GFP mutant and β-arrestin-2-RFP. 48 hours after transfection, the cells were serum starved for one hour prior to treatment with the indicated chemokine at 100 nM for 45 minutes at 37°C. The samples were then washed once with HBSS and fixed in a 6% formaldehyde solution for 30 minutes in the dark at room temperature. Cells were then washed four times with PBS and subsequently imaged with a Zeiss CSU-X1 spinning disk confocal microscope using the corresponding lasers to excite GFP (480 nm) and RFP (561 nm). Confocal images were arranged and analyzed using ImageJ (NIH, Bethesda, MD).
3
CXCR3 and β-Arrestin 2 Interaction Assay
4
Chemokine-Induced Dynamics in HEK293 Cells
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HEK293 cells were plated on rat-tail-collagen-coated 35 mm glass-bottomed dishes (MatTek Corporation, Ashland, MA) and transiently transfected using PEI with the listed constructs. Forty-eight hours following transfection, the cells were washed once with PBS and then serum starved for 1 h. The cells were subsequently treated with a control of serum-free media or the listed chemokine at 100 nM or VUF10661 at 10 µM for 45 min at 37 °C. Following stimulation, the cells were washed once with HBSS and fixed at room temperature in the dark in a 6% formaldehyde solution for 20 min. Cells were subsequently washed four times with PBS and then imaged. The cells were imaged with a Zeiss CSU-X1 spinning disk confocal microscope using the corresponding lasers for GFP (480 nm excitation), RFP (561 nm excitation), and mCerulean (433 nm excitation). Images were analyzed using ImageJ 2.3.0 (NIH, Bethesda, MD).
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