Cytotoxicity Assay for Bacterial Strains

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.

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Tietze E., Dabrowski P.W., Prager R., Radonic A., Fruth A., Auraß P., Nitsche A., Mielke M, & Flieger A. (2015). Comparative Genomic Analysis of Two Novel Sporadic Shiga Toxin-Producing Escherichia coli O104:H4 Strains Isolated 2011 in Germany. PLoS ONE, 10(4), e0122074.

Publication 2015

TSB DMEM CytoTox96 Non-Radioactive Cytotoxicity Assay

Assay Bacterial Culture cell Cytotox96 Promega Radioactive Strains Vero cell

Corresponding Organization :

Other organizations : Robert Koch Institute

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Variable analysis

independent variables

Bacterial strains grown to exponential phase in TSB (Difco)

dependent variables

LDH release in Vero cell monolayers after 48h of incubation

control variables

Equal growth of bacterial cultures confirmed by OD600 readings
Washed confluent Vero cell monolayers in 100ul DMEM/10% FCS in 96 well plates

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