Fetal calf serum (fcs)

Primary mouse macrophages (BMDMs) were differentiated for 6 d and cultured for up to 9 d in DMEM (Gibco) supplemented with 10% FCS (Bioconcept), 20% 3T3 supernatant (MCSF), 10% Hepes (Gibco), and 10% nonessential amino acids (Gibco). Immortalization of macrophages was performed as previously described (Blasi et al, 1985 (link); Broz et al, 2010 (link)). Immortalized macrophages (iBMDMs) were cultured in DMEM complemented with 10% FCS (Bioconcept), 10% MCSF (3T3 supernatant), 10% Hepes (Amimed), and 10% nonessential amino acids (Life Technologies). To passage the BMDMs and iBMDMs, the cells were washed with PBS and left to detach at 4°C for 15 min and scarped using cell scrapers (Sarstedt), spun down at 300g for 5 min at 4°C, and resuspended in the appropriate amount of medium.

Primary wild-type BMDMs from C57BL/6JRj mice (Janvier) were differentiated in DMEM (Sigma) with 20% M-CSF (supernatants of L929 mouse fibroblasts), 10% v/v FCS, 10 mM HEPES, nonessential amino acids and penicillin (100 IU ml⁻¹)/streptomycin (100 μg ml⁻¹) (all BioConcept). One day before infection, BMDMs were seeded into 24- or 96-well plates (Eppendorf) at a density of 1.5 × 10⁵ or 5 × 10⁴ cells per well in DMEM (Sigma) with 10% M-CSF (supernatants of L929 mouse fibroblasts), 10% v/v FCS, 10 mM HEPES and nonessential amino acids (all BioConcept). Where required, BMDMs were pre-stimulated overnight with LPS (from Escherichia coli strain O111:B4 (InvivoGen; tlr-3pelps)). F. novicida were grown overnight at 37 °C with aeration as described above. The bacteria were added to the BMDMs at a multiplicity of infection of 100 or the indicated value. The plates were centrifuged for 5 min at 500g to ensure similar adhesion of the bacteria to the cells and were incubated for 120 min at 37 °C. Next, the medium was replaced with fresh medium containing 10 μg ml⁻¹ gentamicin (BioConcept) to kill extracellular bacteria, then plates were incubated at 37 °C for the indicated length of time.

LLC-MK2 cells (Rhesus monkey kidney cell line, provided by IZSLER, Brescia, Italy) were grown in antibiotic-free growth medium consisting of 500 ml Eagle’s minimum essential medium (EMEM, Gibco, Thermo Fisher Scientific, Invitrogen, Carlsbad, CA, United States) supplemented with 10% heat-inactivated fetal calf serum (FCS, BioConcept, Allschwil, Switzerland), 5 ml L-glutamine (Gibco, Thermo Fisher Scientific), and 6 ml glucose (0.06 g/ml; Sigma-Aldrich Co., St. Louis, MO, United States). Following infection, growth medium was replaced by Chlamydia cultivation medium consisting of 500 ml EMEM supplemented with 20% FCS (BioConcept), 5 ml L-glutamine (Gibco, Thermo Fisher Scientific), and 2 g glucose (Sigma-Aldrich) with or without 0.7 ml cycloheximide (1 mg/ml; Sigma-Aldrich) as described (Donati et al., 2010 (link); Wanninger et al., 2016 (link)).