To measure β-arrestin 2 recruitment, HEK293 cells were transiently transfected with either wild-type GIPR, R190Q, E288G or R190Q;E288G and the donor Rluc8-Arrestin-3-Sp1, the acceptor mem-linker-citrine-SH3 and GPCR kinase 2 (GRK2) to facilitate β-arrestin 2 recruitment. Two days after transfection, the cells were washed with PBS and re-suspended in PBS with 5 mmol/L glucose. Subsequently, 85 μL of the cell suspension solution was transferred to its respective wells on a white 96-well isoplate followed by the addition of PBS with 5 μmol/L coelenterazine-h. After a 10 min incubation of the cells with coelenterazine-h, increasing concentration of endogenous GIP(1-42) were added and luminescence was measured by the Berthold Technologies Mithras Multilabel Reader (Rluc8 at 485 ± 40 nm and YFP at 530 ± 25 nm).
Mithras multilabel reader
Manufactured by Berthold Technologies
The Mithras Multilabel Reader is a versatile laboratory instrument designed for high-throughput measurements. It is capable of detecting and quantifying various types of luminescent, fluorescent, and absorbance signals in microplate samples.
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2 protocols using mithras multilabel reader
1
Measuring GIPR β-Arrestin 2 Recruitment
2
Evaluating GIPR Variant Effects on cAMP and β-Arrestin 2
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For the cAMP assay, HEK293 cells were transiently transfected with either GIPR wt or GIPR‐[E354Q] and the bioluminescence resonance energy transfer (BRET) Epac‐based sensor for cAMP, CAMYEL (cAMP sensor using YFP‐Epac‐RLuc).33 For the β‐arrestin 2 assay, HEK293 cells were transiently transfected with either GIPR wt or GIPR‐[E354Q] and the donor Rluc8‐Arrestin‐3‐Sp1, the acceptor mem‐linker‐citrine‐SH3 and the GPCR kinase 2, GRK2 to facilitate β‐arrestin 2 recruitment.34 Two days following transfection, the cells were washed with PBS and resuspended in PBS with 5 mmol/L glucose. Then, 85 µL of the cell suspension solution was added to each well of a black‐white 96‐well isoplate followed by the addition of PBS with 5 µmol/L coelenterazine‐h. Following a 10‐minute. incubation, increasing concentrations of GIP(1‐42) were added and incubated for an additional 30 minutes. Luminescence was measured by the Berthold Technologies Mithras Multilabel Reader (Rluc8 at 485 ± 40 nm and YFP at 530 ± 25 nm).
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