Chlamydia Cultivation in LLC-MK2 Cells

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)).