384 well plate

384-well plates (PerkinElmer) were coated with growth factor-reduced Matrigel (Corning) and incubated for 30 min at 37°C. ECGM-MV2 containing 4500 HDLECs (25 μl per well) was added and incubated for 60 min at 37°C. Macrophage media containing 2250 human iPS-derived macrophages-RFP was then added (25 μl per well). For time-lapse imaging acquisition, an Evos FL Auto Cell Imaging System (Thermo Fisher Scientific) was used and one image was taken every 8 min for 15 h. At the end of image acquisition, cells were fixed with 4% PFA solution (Santa Cruz Biotechnology) for 30 min and washed with PBS for further analysis (e.g. immunostaining).

LNT-229 cells were seeded on 384 Well Plates (Perkin Elmer, Waltham, MA, USA) and fixed with 4% paraformaldehyde. Cells were permeabilized with 0.5% Triton-X 100 (VWR, Darmstadt, Germany) in PBS (10 min) followed by blocking with 1% BSA in PBS (Gibco) for 1 h. Primary (anti-LAMP2, ab25631, Abcam); anti-LC3B (Cell Signaling #2775); anti-p62 (mouse antibody, M162-3, MBL International, Woburn, MA, USA)) and secondary antibodies as well as nuclear and cytoplasmic staining reagents (Alexa Fluor-coupled antibodies (Life Technologies); DRAQ5 (Cell Signaling); HSC Cell Mask Deep red stain (Life Technologies)) were incubated in 0.1% BSA in PBS for 1 h with three washes of PBS in between. Images were acquired on PerkinElmer's Opera High Content Screening System with a 60× water-immersion objective and visualized with Acapella High Content Imaging Analysis software (Perkin Elmer). The number of antibody-labeled spots per cell were automatically counted using Acapella software.

Jurkat human T-cell line (ATCC, clone E6.1) was exposed to a range of MALT1i concentrations and assessed for viability and inhibition of cytokine expression following cell activation. Cells were cultured in RPMI/10% FBS (Thermofisher, Waltham, MA) and maintained under a concentration of 3 x 10⁶ cells/ml. MALT1i at different concentrations were stamped by ECHO onto 384-well plates (PerkinElmer, Waltham, MA) following which cells were plated in fresh media and incubated for 30 min before stimulation with soluble α-CD3/-CD28/-CD2 (ImmunoCult, Stemcell Technologies, Vancouver, Canada) for 24 h. Supernatants were collected and processed immediately for cytokine analysis or stored at -80°C. To assess viability of cells treated with compound, cells were lysed with CTG reagent (Promega, Madison, WI), and measured by luminometer.

Cells were fixed in 4% paraformaldehyde or 100% ice‐cold methanol at −20°C (for TAX1BP1 staining), permeabilized with 0.1% Triton X‐100 in PBS and blocked with 3% bovine serum albumin in PBS with 0.1% Triton X‐100 and 0.1% saponin. Indirect immunofluorescence staining was followed by mounting in ProLong Gold (ThermoFisher Scientific). Confocal laser scanning microscopy was performed on a TCS SP5 AOBS system equipped with standard PMT detectors as well as sensitive HyD detectors, a 63×/1.4 NA oil immersion objective or an HC PL APO 20×/0.7NA dry objective. Lasers used were HeNe 633 nm, DPSS 561 nm, Ar 488 nm and Diode 405 nm. Acquisition and hardware was controlled by LAS AF software (Leica Microsystems).
Images were processed using Fiji software (https://imagej.net/Fiji), Adobe Photoshop, and Illustrator. Automated quantifications were done with Cell Profiler software 44. Graphs and statistical analysis were done using Excel (Microsoft Corporation) or GraphPad Prism (GraphPad Software).
For the siRNA screen, 384‐well plates (PerkinElmer) were automatically imaged using an Opera automated spinning disk confocal microscope with a UPLAPO 60×/1.2NA water objective. Images were acquired with a High QE CCD camera and analyzed with the Acapella 2.6 Studio software (PerkinElmer).

Stable PDO models were plated in 384-well plates (product number 6007668, PerkinElmer) at approximately 500 cells per well in a 20 µL Geltrex (product number A1413202, Thermo Fisher Scientific) plus 10 µL growth media mix. Organoids were allowed to grow for 3 to 4 days before adding compounds in 10 µL growth media for 72 h. We then added 40 µL of CellTiter-Glo® 3D Cell Viability Assay (product number G9681, Promega) and incubated for 30 min with gentle agitation, protected from light. The luminescence signals were measured with a Varioskan LUX microplate reader (Thermo Fisher Scientific, USA). The dose‒response curves and relative IC50 values were generated by using a dose‒response nonlinear regression model from GraphPad Prism (Dotmatics, USA).

Assays were carried out using the PathHunter hCB₁_bgal, hCB₂_bgal, or mCB₂_bgal CHOK1 β-arrestin recruitment assay kit (DiscoveRx, Fremont, CA, USA) as previously described [28 (link)]. A total of 5000 cells per well were seeded in 384-well plates (Perkin Elmer, MA, USA) containing 20 μL cell culture medium and incubated for 16–18 h at 37 °C and 5% CO₂. Cells were stimulated with 10 μM of each agonist or 11 increasing concentrations and incubated for 90 min at 37 °C and 5% CO₂. In the inverse agonist assays, cells were exposed to 10 μM of RO6851228 or 11 increasing concentrations and incubated for 30 min at 37 °C and 5% CO₂, followed by the addition of the EC₈₀ concentration of CP55940 (25 nM for CHOK1hCB₁_bgal and 46 nM for CHOK1hCB₂_bgal). The cells were incubated for 90 min at 37 °C and 5% CO₂. Compounds in DMSO stock solutions were added using a HP D300 Digital Dispenser (Tecan, Mannedorf, Switzerland). The final concentration of organic solvent per assay point was ≤ 0.1%. The PathHunter Detection mixture was used, according to the manufacturer’s protocol, to determine the β-galactosidase enzyme activity. Detection mixture, 12 μL per well, was added and the plate was incubated for 1 h in the dark at room temperature. Chemiluminescence, indicated as relative light unit, was measured on an EnVision multilabel plate reader (Perkin Elmer, MA, USA).