Glutagro supplement

One week after the last vaccine dose, we euthanized mice and removed the spleen and lungs to assess immune responses. Single-cell suspensions of splenocytes were prepared by gently pressing the cells out of the spleen sac; lysing red blood cells with PharmLyse (BD Pharmingen); washing the cells; and filtering through a 70 µm nylon cell strainer (Falcon). Single-cell suspensions of lung cells were prepared by cutting the lung into small pieces with a scalpel; incubating at 37°C for 1 h with shaking in 10 mL of digestion solution (300 U/mL collagenase type II [Worthington] and 0.15 mg/mL DNase I [Worthington] in PBS); filtering through a 40-µm nylon cell strainer (Falcon); lysing red blood cells with PharmLyse (BD Pharmingen); and washing the cells. Advanced RPMI-1640 (Invitrogen) supplemented with 2% heat-inactivated fetal bovine serum, 2 mM glutamine dipeptide (glutaGRO Supplement, Corning), 10 mM HEPES buffer, 50 µM β-mercaptoethanol, and penicillin (100 IU/mL)-streptomycin (100 µg/mL) was used as the medium.

Murine RAW 264.7 macrophages (ATCC TIB-71) were grown at 37°C in a humidified atmosphere containing 5% CO₂ in RPMI 1640 medium supplemented with 5% fetal bovine serum, 1 mM sodium pyruvate solution, and 2 mM GlutaGro supplement (Corning). HepG2 cells (ATCC HB-8065) were cultured in Dulbecco-modified Eagle medium (DMEM), 10% fetal bovine serum (FBS), and 1× penicillin streptomycin solution (100 U/ml). M. tuberculosis H37Rv-LP (ATCC 25618) constitutively expressing codon-optimized DsRed from plasmid pBlazeC8 (DREAM8) (Carroll et al., 2018 (link)) was cultured at 37°C in Middlebrook 7H9 medium containing 10% v/v OADC (oleic acid, dextrose, catalase) supplement (Becton Dickinson) and 0.05% w/v Tween 80 (7H9-Tw-OADC) plus 100 μg/ml hygromycin B.