Intracellular Bacterial Quantification in Macrophages

Bone marrow derived macrophages were infected with the indicated multiplicity of infection (MOI) of Fn or SchuS4 as previously described (Griffin et al., 2013 (link)). Briefly, bacteria were diluted to the indicated MOI and added to BMM. BMM were incubated for 90 min at 37^∘C/5% CO₂. Then, bacteria containing medium was pipetted off and BMM were incubated with gentamicin (50 μg/ml) for 45 min. BMM were washed extensively with PBS and incubated in DMEM supplemented with 10% heat inactivated fetal bovine serum, L-glutamine, non-essential amino acids, and HEPES (cDMEM; all from Life Technologies). Intracellular bacteria were enumerated by lysing BMM with water and plating lysates on MMH agar as previously described (Bauler et al., 2011 (link)). Where indicated cells were pretreated with 3 mM N-aceytlcysteine (NAC; Sigma), 1 mM L-NMMA (Cayman Chemicals, Ann Arbor, MI, USA) or 250 μM mitoTempo (Enzo Life Sciences, Farmingdale, NY, USA) 16, 24, or 1 h prior to infection, respectively. Effective concentrations of NAC and L-NMMA used in this study were determined by their ability to inhibit production of ROS and RNS in BMM treated with LPS + IFN-γ (Crane, unpublished data). Various concentrations of mitoTempo were tested in BMM. Only concentrations of 250 μM and above had any effect on ROS production in cells (Bauler, unpublished data).