Sodium deoxycholate monohydrate

MycoFluor™ mycoplasma detection kits were purchased from Thermo Scientific (Waltham, MA, USA). The fetal bovine serum was from Gibco (Gibco, Sigma Aldrich Company Ltd., Waltham, MA, USA). Sodium dodecyl sulfate, Sodium Deoxycholate Monohydrate, Triton™ X-100 solution, Glutaraldehyde and Formaldehyde solution, Isopropyl and Ethyl Alcohol, Accutase cocktail, fluorescent dyes Calcein AM, Propidium iodide and Bisbenzimide Hoechst 33342, Culture media alfa-MEM and DMEM, L-glutamine, gentamicin sulfate, Alcian Blue 8GX, Biebrich scarlet, Fast Green FCF, Schiff’s reagent and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA or Gibco, Sigma-Aldrich Company Ltd., Waltham, MA, USA). Dioxidine^® (Hydroxymethylquinoxalindioxide, 10 mg/mL) was purchased from JSC “Valenta Pharm” (Moscow, Russia). Mayer’s hematoxylin, eosin H, Alizarin red S, toluidine blue, neutral buffered formalin, and other reagents for histological analysis were purchased from Labiko LLC (Saint Petersburg, Russia). All other reagents used were of analytical reagent quality.

LGs were decellularized as described previously.²⁶ (link) In brief, LGs were cut into pieces 3 mm in diameter and washed in cold PBS (Sigma-Aldrich, St. Louis, MO, USA) containing 5% penicillin/streptomycin (P/S; Sigma-Aldrich) overnight. Cellular components were removed by incubation in a 1% (w/v) solution of sodium deoxycholate monohydrate (Sigma-Aldrich) for 36 hours with three changes, followed by DNase solution (200 U/mL in PBS; Roche, Basel, Switzerland) for 24 hours, and then washing in PBS + 5% P/S for an additional 24 hours. All incubation steps were performed at 4°C under continuous agitation with interposed washing in PBS + 5% P/S. The decellularized LGs were stored at −80°C until further use.

The bacterial strains and the plasmids used in this study are listed in Supplementary Table 3. The S. enterica serovar Typhimurium strains were the wild-type strain ATCC14028s^{21 (link)} and its ∆ramR mutant (which was produced as described previously)^{22 (link)}. Bacterial strains were grown at 37 °C in Luria–Bertani broth supplemented, when appropriate, with ampicillin (100 µg/ml), kanamycin (25 µg/ml) or chloramphenicol (25 µg/ml).
In order to investigate the effects of the bile acids on ramA expression, 25.6 mg/ml of a crude ox bile extract purchased under the label ‘sodium choleate’ (Sigma-Aldrich, S9875), or 5 mM sodium cholate hydrate, sodium chenodeoxycholate or sodium deoxycholate monohydrate (C6445, C8261 and D5670, respectively, and all from Sigma) was added to the medium. sodium choleate, whose precise composition was not determined, has previously been used in Salmonella gene expression experiments at concentrations of 3% (w/v), i.e., 30 mg/mL or higher^{8 (link)}. We chose a concentration of 25.6 mg/mL, because it was the highest concentration that allowed the normal growth of the Salmonella strains used in this study.