If005

Ct sensitivities to interferon-gamma was assayed as previously described (Walsh et al., 2022 (link)). Briefly, A549 cells were seeded in black 96-well clear-bottomed plates (Corning). The next day, cells were stimulated with 0 U/mL or 100 U/mL interferon gamma (IFNγ; Millipore, IF005) in DMEM supplemented with L-tryptophan (100 µg/mL). After 20 hr, cells were infected in technical duplicate with indicated Chlamydia strains at an MOI of 2. At 24 hr post-infection, plates were fixed with cold 4% PFA in PBS for 20 min. Samples were nuclear stained with Hoechst in PBS for 10 min and sealed using an aluminum adhesive (Thermo Fisher). Inclusions and host cell nuclei were imaged and quantified using the CellInsight CX5 High Content Screening platform (Thermo Fisher; CX51110). Relative bacterial infectivities were calculated as the number of inclusions divided by the total number of host nuclei for each sample. Interferon sensitivity was calculated by normalizing the infectivities of each strain to it’s ‘untreated’ (-IFNγ) control and expressed as a percentage.

Bone marrow-derived macrophages (BMDMs) were isolated and differentiated from bone marrow (tibia and femur) of 8–12 weeks old sex-matched mice. BMDMs were differentiated for 7–9 days on Petri dishes (Greiner Bio-One) in DMEM (Sigma-Aldrich) supplemented with 10% FCS (Gibco/Thermo Fisher Scientific), 15% L929 cell-conditioned medium, 2 mM L-glutamine (Sigma-Aldrich), 100 U/ml penicillin, 100 μg/ml streptomycin, (Sigma-Aldrich) and 50 μM β-mercaptoethanol (Gibco/Thermo Fisher Scientific). Cells were treated with recombinant mouse 100 U/ml IFNγ (Millipore, IF005) for the times indicated.