β mercaptoethanol

Bone marrow cells were isolated as described in the leukocyte isolation section. Cells were cultured at 1.5 x 10⁶ cells/ml for 7 days in RPMI 1640 media (Wisent) supplemented with 10% FBS (Wisent), 50 µM β-mercaptoethanol, antibiotic-antimycotic (Wisent) and 100 ng/ml FMS-like tyrosine kinase 3 ligand (FLT3L) (peprotech, Rocky Hill, NJ, USA, catalog no. 250-31L). On day 7, BMDCs were harvested for stimulation.

Spleens were excised, collected in Hank’s balanced salt solution without calcium and magnesium (HBSS) (Wisent, St. Bruno, QC, Canada), and processed individually. Homogenous cell suspensions were prepared by passing organs through a 70 μm cell strainer (BD Biosciences, Mississauga, ON, Canada). Cells were treated with ACK buffer (0.15M NH₄Cl, 1 mM KHCO₃, 0.1 mM Na₂EDTA; pH 7.2), and then washed with HBSS. Splenocytes were resuspended in RPMI supplemented with 10% fetal bovine serum (FBS), 1 mM penicillin/streptomycin (all from Wisent), and 0.5 mM β-mercaptoethanol (Sigma) (complete RPMI, cRPMI).

Bone marrow extracted from C57BL/6 mice was cultured in the presence of 20 ng/mL granulocyte-macrophage colony-stimulating factor (PeproTech) in “complete DC medium” (CDCM) containing RPMI-1640 (Corning) medium with 10% fetal calf serum (FCS) (Hyclone), 2 mM l-glutamine (Wisent), 100 U/mL penicillin-streptomycin (Wisent), and 0.01% β-mercaptoethanol (Gibco). After 8–10 days, DCs were collected, seeded at 2 million cells/mL, and stimulated where indicated with LPS (O111:B4 Sigma-Aldrich), HDM (low endotoxin from Greer Laboratories), curdlan (InvivoGen), Zym (InvivoGen), or Zym-depleted^{39 (link)} (InvivoGen). HDM was labeled with Alexa Fluor 647 in indicated experiments with the Alexa Fluor 647 Protein Labeling Kit (Invitrogen). Glucose-withdrawal experiments used glucose-free RPMI-1640 (Wisent) supplemented with 10% dialyzed FCS (Wisent), 2 mM l-glutamine, 100 U/mL penicillin-streptomycin, and 0.01% β-mercaptoethanol.