Foetal calf serum

At Day 8 post‐MDM culture (24 h post‐BCG‐GFP stimulation), medium and extracellular bacteria were washed off and macrophages were harvested by incubating cells for 10 min at 37°C with Accutase^® (Sigma). For the single positive control for viability dye, a mixture of dead/alive cells was prepared, as per manufacturer's instructions. Next, 50 μL of previously titrated viability dye (Fixable Viability Dye eFluor^® 780, eBiosciences) (1/1000 in D‐PBS) was added to 50 μL of D‐PBS containing the cells. Then, tubes were incubated for 30 min at 2–8°C in the dark. After this, the excess dye was washed off (D‐PBS [cell culture grade, Gibco], 3% heat‐inactivated foetal calf serum [Gibco] and 0.09% sodium azide [Sigma–Aldrich], pH 7.5) and cell pellets were incubated in 50 μL of blocking buffer (0.05 m Tris‐buffered saline, pH 7.4, containing 1% normal bovine serum, 0.1% azide [Sigma–Aldrich] and 2% heat‐inactivated foetal calf serum [Gibco]) for 20 min at 4°C.

Conditionally immortalised human podocytes were obtained from Dr. Moin A. Saleem (University of Bristol, Southmead Hospital, Bristol, UK). Briefly, human podocytes were conditionally immortalised by introducing a temperature-sensitive SV40-T antigen by transfection33 (link). Additionally, the cells were transfected with a human telomerase construct34 (link). Conditionally immortalised murine podocytes were kindly provided by Dr. Peter Mundel (Department of Medicine, Massachusetts General Hospital, Charlestown, MA, USA). These cells proliferate at a permissive temperature (33°C) and enter growth arrest after transfer to a nonpermissive temperature (37°C). When the podocytes reached approximately 70–80% confluence at 33°C, the podocytes were transferred to 37°C for differentiation in a medium without insulin, transferrin or selenium (ITS) for approximately 7 days.
The growth medium for human podocytes consisted of RPMI 1640 medium (HyClone, USA) supplemented with 10% foetal calf serum (Gibco, USA), 1× penicillin-streptomycin, 1 mM L-glutamine and 1× ITS (Invitrogen, Grand Island, NY, USA) at a permissive temperature (33°C). The growth medium for murine podocytes consisted of RPMI 1640 (HyClone, USA) supplemented with 10% foetal calf serum (Gibco, USA), 10 units/ml recombinant mouse interferon-c (Peprotech, USA), 100 U/ml penicillin G and 100 g/ml streptomycin (Invitrogen, USA).