Wallac envision 2104 multilabel reader

CHO-spap-hA_2BAR cells were cultured as a monolayer in 15 cm ø plates to about 90% confluency. Cells were removed from the plates by scraping into 5 mL of phosphate-buffered saline (PBS) and centrifuged for 5 min at 1500 rpm. The resulting pellets were resuspended in ice-cold Tris-HCl buffer (50 mM Tris-HCl, pH 7.4) and homogenized using an Ultra Turrax homogenizer (IKA Werke GmbH & Co.KG, Staufen, Germany). Centrifugation at 31,000 rpm in an Optima LE-80 K ultracentrifuge with Ti-70 rotor (Beckman Coulter, Fullerton, CA) at 4 °C for 20 min, resulted in separation of membranes and cytosolic fraction. Subsequently, pellet was resuspended in 10 mL Tris-HCl buffer, homogenized and centrifuged once again. The final pellet was suspended in assay buffer (50 mM Tris-HCl buffer, 0.1% (w/v) CHAPS, pH 7.4), ADA was added to break down endogenous adenosine, and the homogenization step was repeated. Aliquots were stored at −80°C and the membrane protein concentration was determined by a BCA protein determination assay [26 (link)]. The BCA results were measured in a Wallac EnVision 2104 Multilabel Reader (Perkin Elmer, Groningen, The Netherlands).

Total protein content was determined using the colorimetric Pierce BCA protein assay kit (Thermo Fisher, Scientific, Waltham, MA) in cell pellets, according to the manufacturer’s instructions. Briefly, cells were washed with PBS, collected with trypsin from the cell culture plates and centrifuged to obtain cell pellets, which were then lysed using RIPA lysis buffer (Sigma, Dorset, UK). Absorbance was measured using a Wallac EnVision 2104 Multilabel Reader (Perkin Elmer, Waltham, MA) at 560 nm. Quantification of total protein was determined by interpolation from a standard curve made of 2-fold serial dilutions of a 2 mg/mL bovine serum albumin (BSA) standard solution. Results were calculated using Microsoft Excel and Originlab Origin software.

Receptor expression after transfection was measured in an enzyme-linked immunosorbent assay (ELISA). After 24 h of transfection, HEK293T cells were detached with phosphate-buffered saline (PBS)/EDTA and seeded into a sterile 96-well poly-D-lysine coated plate at a density of 100,000 cells per well and kept under a humidified atmosphere at 37 °C with 5% CO₂. After an additional 24 h, cells were washed with PBS and fixed with 4% formaldehyde for 10 min at room temperature (rt). Cells were washed twice with Tris-buffered saline (TBS) and were blocked with TBS supplemented with 0.1% TWEEN 20 (TBST) and 2% BSA (w/v)) for 30 min at rt while shaking. Subsequently, the cells were incubated with monoclonal M2 mouse anti-FLAG primary antibody (1:4000) for 2 h at rt while shaking. After removal of the antibody, the cells were washed three times with TBST and incubated with the secondary goat anti-mouse HRP-conjugated antibody (1:10,000) for 1 h at rt while shaking. After a final wash with TBS, the cells were treated with 3,3’,5,5’-Tetramethylbenzidine (TMB, Sigma-Aldrich #T0440) in the dark for maximally 10 min at rt to visualize immunoreactivity. The reaction was quenched with 1 M H₃PO_4, and absorbance was read at 450 nm with a Wallac EnVision 2104 Multilabel reader (PerkinElmer).

A three-fold dilution series of mono-Ub, K48-linked di-Ub, K48-linked tetra-Ub, or linear tetra-Ub was premixed with ZUFSP-His₆ (final concentration 100 nM). K63-linked Ub₄-Rh110 (final concentration 750 nM) was added to initiate the reaction and the plate was centrifuged briefly. Assays were performed in technical triplicate in a total volume of 20 µL of 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT, and 1 mg/mL BSA, in a black ProxiPlate-384 Plus F 384-shallow-well microplate (PerkinElmer, catalog number 6008260). Fluorescence was detected on a Wallac EnVision 2104 Multilabel reader (PerkinElmer; excitation 485 nm, emission 535 nm) with reads interspaced by 30 s of shaking. Initial rates (RFU/min) were calculated from the linear increase in Rh110 fluorescence and expressed as a percentage of the maximum rate (without additional Ub chains).