Raw dual cells

RAW-Dual cells (InvivoGen) were plated at 50,000 cells per well in 45 μL of DMEM with 5% HI-FBS from col 2 to 23 in clear flat-bottom 384 well plates (Greiner Bio-One). Cells attached at room temperature for 1 h. Fifty nanoliter of 10 mM modulator libraries were added by JANUS G3 via pintool to experimental wells (cols 3–22) for a final concentration of 10 μM. Following 1 h incubation, 5 μL of PRR agonist was added via a MultiDrop Combi liquid handler (col 3–23). Cells were incubated at 37°C and 5% CO₂ overnight. Twenty hours later, 12.5 μL of QUANTI-Luc Plus was plated in an opaque, white 384-well plate. Five microliter of RAW-Dual cell supernatant was then added via MultiDrop Combi liquid handler before measuring luminescent values on a BioTek Synergy Neo2 plate reader as soon as the plate was completed. QUANTI-Luc Plus contains a stabilizer that reduces signal decay, allowing for comparable values throughout the read. In parallel, 15 μL of 5× concentrated QuantiBlue was added directly to the remaining cell supernatant in the RAW-Dual cell plate. Absorbance values were measured at varying time intervals at 620 nm. These samples were incubated so that the PRR agonist control reported an absorbance signal of approximately 1 A.U.

Raw 264.7 cells (RAW-dual cells from InvivoGen) were cultured in DMEM (with 4.5 g/L glucose, 2 mM L-glutamine, 10% heat-inactivated FBS, 100 µg/mL Normocin and 1% Pen-Strep) at a seeding density of 1.5 × 10⁴ cells/cm². The medium was renewed twice a week. THP-1 cells (THP1-dual cells from InvivoGen) were maintained in RPMI 1640 (with 2 mM L-glutamine, 25 mM HEPES,10% heat-inactivated FBS, 100 μg/mL Normocin, and 1% Pen-Strep). HL-60 cells were cultured in IMEM with 20% FBS.

All fluorescently tagged antibodies, αCD28/CD3 antibodies, ELISA kits, RBC lysis buffer, Foxp3/Transcription Factor Staining Buffer Set, EDTA, and Cell Proliferation Dye eFluor^™ 670 were purchased from Invitrogen. All inhibitors (modulators) were purchased from MedChemExpress. Bovine serum albumin (BSA) was purchased from VWR Life Science. HBSS, DPBS, PBS, DMEM, RPMI 1640, AIM-V medium, FBS, HI-FBS, HEPES, and non-essential amino acid solutions were purchased from Gibco. RAW-Dual cells, RAW Blue cells, QB buffer and reagent, EndoFit Ovalbumin (OVA), ODN 1826 (CpG), and all other TLR agonists were purchased from InvivoGen. Spleen Dissociation Medium and EasySep Mouse T-Cell Isolation Kit were purchased from STEMCELL Technologies. β-Mercaptoethanol was purchased from MP Biomedicals. Cell Activation Cocktail (without Brefeldin A), Recombinant Mouse GM-CSF (carrier-free) (20 ng/mL), and LEGENDplex MU Th1/Th2 Panel (8-plex) Kit were purchased from BioLegend. ProT2 MHC Class II tetramers and Pro5 MHC Class I pentamers were purchased from ProImmune. All plates, unless noted otherwise, were purchased from Thermo Fisher Scientific.