Differentiation and Stimulation of Mouse Bone Marrow-Derived Macrophages

Mouse bone marrow cells were differentiated into macrophages in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) low-endotoxin fetal bovine serum (FBS; Omega Scientific) and 20% (v/v) L929-conditioned medium for 5 days. For stimulation, cells were replated overnight at 1.0 × 10⁵ cells per well in 96-well plates. For inflammasome stimulations, cells were primed with Pam3CSK4 (1 μg ml⁻¹) for 5 h and then stimulated with 10 μg ml⁻¹ nigericin in Opti-MEM I media (Thermo Fisher Scientific). For flagellin or LPS electroporation^{39 (link)}, 1.0 × 10⁶ Pam3CSK4-primed BMDMs were electroporated with 1.0 μg LPS or 0.2 μg flagellin in 100 μl R buffer using a neon 100-μl tip with 1,720 voltage, 10 width, 2 pulse settings. Electroporated cells were added to 990 μl Opti-MEM I medium and cultured for 2 h. BMDMs treated with venetoclax (25 μM for 6 h), doxorubicin (10 μM for 6 h), TNF plus actinomycin D (20 ng ml⁻¹ TNF, 500 ng ml⁻¹ actinomycin D for 6 h), or TNF plus zVAD (100 ng ml⁻¹ TNF, 20 μM zVAD for 16 h) were not primed. For lysis controls, cells were lysed with 0.25% Triton-X100 in medium. Where indicated, BMDMs were cultured with the indicated concentration of anti-NINJ1 clone 25^{1 (link)} (mouse IgG2a; Genentech), anti-NINJ1 clone D1 (mouse IgG2a; Genentech), anti-ninjurin clone 50 (BD Biosciences BD610777, raised against human NINJ1), or an isotype control mouse IgG2a (Thermo Fisher Scientific). Prior to addition to cells, anti-ninjurin clone 50 and isotype control mouse IgG2a antibodies were dialysed against PBS to remove sodium azide using Slide-A-Lyzer MINI Dialysis Device with a 10K MWCO (Thermo Fisher Scientific) according to the manufacturer’s instructions. Synthetic peptides used were mouse NINJ1 amino acids 26–37 (PPRWGLRNRPIN, Genentech) and its sequence-scrambled analogue (PWPPRRNRNGLI, Genentech). BMDMs were pre-treated with antibody or peptide for 10 min prior to addition of treatment.