Natriumpyruvate

Spleens or lungs were aseptically harvested from euthanized mice. Lungs were first homogenized in Gentle MACS tubes C (Miltenyi Biotec) or chopped into small pieces using scalpels, followed by 1 h of collagenase digestion (Sigma-Aldrich; catalog no. C5138) at 37°C and 5% CO₂. The lung homogenate and spleens were forced through 70- to 100-μm cell strainers (BD Biosciences) with the stopper from a 5-ml syringe (BD) and washed twice with cold RPMI medium (Gibco; RPMI 1640) by centrifuging 5 min at 1,800 rpm. A red blood cell lysis step was performed in between washes (Roche, catalog no. 11814389001). Cells were finally resuspended in enriched RPMI medium (RPMI 1640, 10% heat-inactivated fetal calf serum (FCS) (Biochrom GmbH), 10 mM HEPES (Invitrogen), 2 mM l-glutamine (Invitrogen), 1 mM Natriumpyruvate (Invitrogen), 1× nonessential amino acids (MP Biomedicals, LLC), 5 × 10⁵ M 2-mercaptoethanol (Sigma-Aldrich), and penicillin-streptomycin (Gibco). Cells were counted using an automatic Nucleocounter (Chemotec) and adjusted to 2 × 10⁵ cells/well for enzyme-linked immunosorbent assay (ELISA) and 1  × 10⁶ to 2 × 10⁶ cells/well for flow cytometry.

Spleens or lung were aseptically harvested from euthanized mice. Lungs were first homogenized in Gentle MACS tubes C (Miltenyi Biotec) followed by 1 h of collagenase-digestion (Sigma Aldrich; C5138) at 37 degrees, 5% CO₂. The lung homogenate and spleens were forced through 100 µm cell strainers (BD Biosciences) with the stopper from a 5 ml syringe (BD) and washed twice with cold RPMI medium (Gibco; RPMI-1640) by centrifuging 5 min at 1,800 rpm. Cells were finally re-suspended in enriched RPMI medium [RPMI-1640, 10% heat-inactivated FCS (Biochrom Gmbh), 10 mM Hepes (Invitrogen), 2 mM L-Glutamine (Invitrogen), 1 mM Natriumpyruvate (Invitrogen), 1× Non-essential amino acids (MP Biomedicals, LLC), 5×10^-5 M 2-mercaptoethanol (Sigma-Aldrich), and PenStrep (Gibco)]. Cells were counted using an automatic Nucleocounter™ (Chemotec) and adjusted to 2x10⁵ cells/well for ELISA and 1-2x10⁶ cells/well for flow cytometry.

Single splenocyte suspensions were prepared by homogenisation through 100 μm cell strainers followed by washing in RPMI 1640 (Invitrogen) and adjustment to 5 × 10⁶ splenocytes per 600 μl in MGIA media. MGIA media were either standard media (RPMI-1640, 10% heat-inactivated FCS (Biochrom Gmbh) + 10 mM Hepes (Invitrogen) + 2 mM L-Glutamine (Invitrogen))¹⁵ or enriched media (standard media + 1 mM Natriumpyruvate (Invitrogen) + 1 × Non-essential amino acids (MP Biomedicals, LLC) + 5 × 10⁻⁵M 2-mercaptoethanol (Sigma-Aldrich)). The cell suspensions were cultured in 2 ml screw cap tubes (Sarstedt) on a 360° tube rotator (Intelli-Mixer Rm-2 L, ELMI) or in a rack at 37 °C for four days. At different time points, the splenocytes were counted with an automatic Nucelocounter^TM (Chemotec) or manually using Nigrosine. All cell work pre-M.tb infection was done in BSL2.