Proxiplate 384 plus

cAMP assays were performed using a homogeneous time-resolved fluorescence (HTRF®) cAMP dynamic kit (CisBio Bioassays, Codolet, France). Cells were detached by incubation at 37°C for 5–10 min with Versene (Invitrogen, Paisley, U.K.), counted and added at 5000 cells/well to low-volume 384-well plates (Proxi-plate™ 384 Plus, PerkinElmer Life Sciences). cAMP production was stimulated by the addition of varying concentrations of secretin followed by a 30 min incubation at room temperature. Outputs were measured by using a PHERAstar FS plate reader (BMG Labtech Ltd, Buckinghamshire, U.K.).

Fragment inhibitory activity on Mac1 was assessed by the displacement of an ADPr-conjugated biotin peptide from the His₆-tagged Mac1 using HTRF with an Eu³⁺-conjugated anti-His₆ antibody donor and streptavidin-conjugated acceptor. Compounds were dispensed into white ProxiPlate-384 Plus (PerkinElmer) assay plates using an Echo 525 liquid handler (Labcyte). Binding assays were conducted in a final volume of 16 μl with 12.5 nM Mac1, 400 nM peptide ARTK(Bio)QTARK(Aoa-RADP)S [synthesized by Cambridge Peptides (Birmingham, UK)], 1:125 streptavidin-XL665 (Cisbio), and 1:20,000 anti–His₆-Eu³⁺ cryptate (PerkinElmer) in assay buffer [25 mM Hepes (pH 7.0), 20 mM NaCl, 0.05% bovine serum albumin, and 0.05% Tween 20]. Assay reagents were dispensed into plates using a Multidrop combi (Thermo Fisher Scientific) and incubated at room temperature for 1 hour. Fluorescence was measured using a PHERAstar microplate reader (BMG) using the HTRF module with dual-emission protocol (A = excitation of 320 nm, emission of 665 nm and B = excitation of 320 nm, emission of 620 nm). Raw data were processed to give an HTRF ratio (channel A/B × 10,000), which was used to generate IC₅₀ curves by nonlinear regression using GraphPad Prism v8 (GraphPad Software, CA, USA).

The Monoamine oxidase (MAO) activity measurements were performed using a MAO-Glo assay kit (Promega, Madison, WI), in a 384-well plate (Proxiplate 384 Plus, Perkin Elmer, Waltham, MA) with the miniaturization of final assay volume to 20 µL, as per the manufacturer’s instructions. In brief, MAO reactions were terminated after a 60-min enzymatic reaction, by the addition of reconstituted Luciferin Detection Reagent. Following a 20-min incubation with the detection reagent, the luminescence was measured using a multi-label plate reader (Envision, Perkin Elmer). IC₅₀ values were estimated by fitting percentage inhibition vs. compound concentration in a variable slope (four parameter) fit.

Assays were performed as described previously (33 (link)) with minor modifications from the the manufacturer’s protocol (PerkinElmer). All reagents were diluted in 25 mM Hepes, 100 mM NaCl, 0.1% bovine serum albumin (pH 7.4) supplemented with 0.05% CHAPS and allowed to equilibrate to room temperature before addition to plates. An 11-point 1:2.5 serial dilution of the ligands was prepared over the range of 5000 to 0 μM, and 0.1 μl was transferred to low-volume 384-well plates filled with 5 μl of the assay buffer (ProxiPlate-384 Plus, PerkinElmer), followed by 7 μl of biotinylated peptide H-ALREIRRYQK(ac)STELLIRKLK(biotin)-OH and His-tagged protein to achieve a final assay concentration of 50 nM. Plates were sealed and incubated for a further 30 min before the addition of 8 μl of the mixture of streptavidin-coated donor beads (12.5 μg/ml) and nickel chelate acceptor beads (12.5 μg/ml) under low light conditions. Plates were foil-sealed to protect from light, incubated at room temperature for 60 min, and read on a PHERAstar FS plate reader (BMG Labtech) using an AlphaScreen 680 excitation/570 emission filter set. IC₅₀ (median inhibitory concentration) values were calculated in Prism 5 (GraphPad Software) after normalization against corresponding dimethyl sulfoxide (DMSO) controls and are given as the final concentration of the compound in the 20-μl reaction volume.

Unless otherwise noted, experiments were performed in white, 384-well microtiter plates (Corning 3572 or PerkinElmer ProxiPlate-384 Plus) in 30 μL or 10 μL assay volume, respectively. TR-FRET measurements were acquired on a Tecan SPARK plate reader with SPARKCONTROL software version V2.1 (Tecan Group Ltd.), with the following settings: 340/50 nm excitation, 490/10 nm (Tb), 520/10 nm (FITC) emission, 100 μs delay, and 400 μs integration. The 490/10 and 520/10 nm emission channels were acquired with a 50% mirror and a dichroic 510 mirror, respectively, using independently optimized detector gain settings unless specified otherwise. The TR-FRET ratio was taken as the 520/490 nm intensity ratio on a per-well basis.

Macrophage precursors were seeded at 4 × 10⁴ cell/well in optically-clear bottom CellCarrier 96-well plates (Perkin Elmer), and differentiated in macrophage media for a week. For detection of pSYK, the pSYK/tSYK HTRF kits (Cisbio) were used, according to manufacturer’s instructions. Cells were stimulated with goat polyclonal human TREM2 antibody (AF1828, R&D systems) for 5 min at 37 °C. Normal goat IgG (R&D Systems) was used as control. Medium was aspirated and cells were lysed in supplemented lysis buffer (Cisbio), placed on an orbital shaker for 20 min at room temperature. Experiments were run with 3 replicate wells. Cell lysates were dispensed into a ProxiPlate-384 Plus (Perkin-Elmer), followed by pre-mixed Eu³⁺-cryptate and d2 antibody diluted in detection buffer. The plate was sealed and incubated overnight at RT, then read on a PHERAstar FSX (BMG Labtech) using a HTRF optic to detect emission at 665 nm and 620 nm. Data was exported as two RFU values, and signal/noise ratio was calculated according to manufacturer’s instructions.