Tryptic soy broth (tsb)

Toxicity towards Vero cells was determined as described previously [44] (link) with modifications. Briefly, strains were grown to exponential phase in TSB (Difco), then diluted 1∶100 in 5 ml TSB, and incubated for 20 h at 37°C with agitation (180 rpm). Next, 100 ul of 8-fold to 512-fold DMEM (GE Healthcare) diluted cell free culture supernatants of the TSB-grown strains were added to washed confluent Vero cell monolayers in 100 ul DMEM/10% FCS in 96 well plates in triplicates. For each experiment fresh culture supernatants were produced and equal growth of the bacterial cultures was confirmed by OD600 readings. After 48 h of incubation at 37°C, supernatants were analyzed for LDH release by means of the CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega) according to the manufacturer’s protocol. All values shown are corrected by the background reading of the diluted culture supernatants. In all assays, 256-fold diluted E. coli culture supernatants were determined as best discriminative and are therefore shown.

Toxicity towards Vero cells was determined as described previously [17 (link)]. Briefly, strains were grown to exponential phase in TSB (Difco), then diluted 1:100 in 5ml TSB and incubated for 20h at 37°C with agitation (180rpm). Next, 100ul of 8-fold to 512-fold DMEM (GE Healthcare) diluted cell free culture supernatants of the TSB-grown strains were added to washed confluent Vero cell monolayers in 100ul DMEM/10% FCS in 96 well plates in triplicates. For each experiment fresh culture supernatants were produced and equal growth of the bacterial cultures was confirmed by OD600 readings. After 48h of incubation at 37°C, supernatants were analyzed for LDH release by means of the CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega) according to the manufacturer’s protocol.