Cell Culture and Bacteria

HeLa (ATCC^® CCL-2.1™) and THP-1 (ATCC^® TIB-202™) cells were grown in RPMI1640 medium (Thermo Fisher Scientific, Dreieich, Germany) supplemented with 10% FCS (Sigma/Merck, Darmstadt, Germany). For differentiation of THP-1 cells into macrophages, 5 × 10⁵ cells were seeded into a 12-well plate and treated with 20 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma/Merck, Darmstadt, Germany) for 72 h. HeLa cells with a knockout of Bax and Bak or overexpressing Bcl-XL were a kind gift from A. Weber and were generated as described before (Weber et al., 2016; Brokatzky et al., 2019). For S. negevensis preparation HeLa cells were grown to 50-60% confluence and infected in infection medium (RPMI w/o HEPES supplemented with 5% heat inactivated FCS) at MOI 1 for 6 h at 35°C, 5% CO₂. Medium was then replaced by fresh infection medium and infected cells were grown for 3 days. Cells were mechanically detached, and bacteria released using ~ 2–5 mm glass beads (Carl Roth, Karlsruhe, Germany). Low speed supernatant (600 × g) was subjected to high-speed centrifugation (20,000 × g) to pellet bacteria. Bacteria were washed with 5 ml SPG buffer [250 mM sucrose, 4 mM monopotassium phosphate, 10 mM disodium phosphate, and 5 mM glutamate (pH 7.4)], aliquoted and stored at -80°C in the SPG buffer. Work with S. negevensis was conducted in a biosafety level 2 laboratory registered with the Government of Lower Franconia under code 55.1-8791.1.30.