Purification and Use of C. difficile Toxins

TcdA and TcdB from C. difficile NAP1/027 strain were purified as described previously from 72 h culture supernatants using dialysis culture in Brain Heart Infusion broth^{47 (link),48 (link)}. TcdB from C. difficile VPI 10463 was a generous gift from Klaus Aktories (University of Freiburg, Germany) and purified as described before.^{49 (link)}Amiodarone hydrochloride was ordered from Thermo Fisher Scientific (Waltham, USA; J60456) or from Supelco (Bellefonte, USA; PHR1164), dissolved in dimethyl sulfoxide (DMSO) to obtain a 10 mM stock solution, which was stored at -20°C. Bafilomycin A1 (sc-201550; 100 µM in DMSO), inositol hexakisphosphate (InsP6; sc-250718; stock: 100 mM in H₂O) and castanospermine (sc-201358; stock: 100 mM in H₂O) were from SantaCruz Biotechnology (Dallas, USA). N-ethylmaleimide (NEM) and quinacrine were ordered from SigmaAldrich. LysoTracker™ Green DND-26 and Hoechst 33342 were obtained from Thermo Fisher Scientific.