Assessing C. jejuni Impact on HT-29/B6 Viability

To study effects of C. jejuni on HT-29/B6 viability, Calcein-acetoxymethyl/Hoechst staining was performed. Calcein-acetoxymethyl (Calcein-AM; Biotium) has already been described as a green fluorescent indicator of viable cells in cytotoxicity assays [29 (link), 30 (link)]. Hoechst 33342 (Thermo Fisher Scientific) (hereinafter referred to as Hoechst) is used as a blue fluorescent marker of the nuclei of all cells [53 (link)]. Viability of HT-29/B6 cells was investigated in 96 well plates (Sarstedt). After 7 days differentiation, HT-29/B6 with ~ 80–90% confluence were infected with wild type, ::Cj1492c and ::Cj1507c for 5 h and 24 h as described before. Infected monolayers were washed 3 times with PBS and incubated with 0.4 µM Calcein-AM at 37 ℃ with 5% CO₂ for 30 min. 5 µg/ml Hoechst was added to each well and incubated 15 min at room temperature followed by two washes with PBS. Fluorescence micrographs were subsequently captured with a Leica DMI6000 (Leica). The relative viability of host cells was calculated as percentage of the number of viable cells compared to the total cell numbers automatically determined using the ImageJ software as described above [27 (link)]. Presented results are calculated from three individual assays.