4 6 diamidino 2 phenylindole (dapi)

The bacterial suspension was centrifuged (3200× g, 10 min, 4 °C) in conic glass tubes. Bacteria were treated with 2% formaldehyde [stock: 8% formaldehyde pH 7, diluted with PBS (6 mM Na₂HPO₄, 1.8 mM NaH₂PO₄, and 145 mM NaCl with bi-distilled water, pH 7)] for 30 min. The bacteria were centrifuged and resuspended in 70% ethanol for further fixation and long-term storage at −20 °C.
After a minimum of one day at −20 °C, the samples were stained with 0.24 µM 4′,6-di-amidino-2-phenyl-indole (DAPI, Sigma-Aldrich, St-Louis, USA) overnight, according to Koch et al. [31 (link)]. The measurement was performed according to Gelder et al. [34 (link)], but with a different neutral density filter (ND 2.6) for the side scatter (SSC) and measuring 250,000 cells in the cell gate (Supplementary Material, Figure S1). Raw cytometric data can be found at www.flowrespository.org with the flow repository ID: FR-FCM-Z24C.
For flow cytometric analysis and statistical data analysis, FlowJo V10 (FlowJo, LLC, Ashland, USA) was used to visualize each sample in 2D plots using forward scatter (FSC) vs. DAPI fluorescence. The relative cell abundance per gate was exported as .txt and jointly evaluated in R (vegan package) [35 ].

To determine at which concentrations SM become toxic to RH30 and NK cells, the cells were challenged with increasing doses of SM, harvested, and stained with 4′,6-diamidino-2-phenylindole (DAPI; BioLegend, USA) after 24 and 48 h. The cell suspensions were measured by flow cytometry using a BD FACSCanto10c™ instrument (BD Bioscience, San Diego, CA, USA) and data were analyzed using FlowJo (FlowJo LLC, Ashland, Oregon, USA), first gating on single cells, then defining the DAPI negative population as viable cells. To analyze the effect of SM on the proliferation of NK cells, the cells were isolated and taken into culture as described above with increasing doses of SM in addition to IL-2. On days 0, 3, 6, and 10, NK cells were harvested and counted using the COULTER^® Ac·T diff™ Analyzer (Beckman Coulter, Germany), an automatic cell counter.