Sodium lauroyl sarcosinate

Outer membrane proteins were isolated as previously described (Huang et al., 2006 (link)). Bacteria were grown overnight, diluted at 1:100 into fresh TSB medium without antibiotics and then harvested at OD₆₀₀ = 1.0 by centrifugation at 4000 g for 15 min at 4°C. After being washed three times with sterile saline buffer (0.85% NaCl), the bacterial pellet was resuspended in 10 mL sonication buffer (50 mM Tris-HCl, pH 7.4). The solution was sonicated intermittently for periods of 7 s interspersed at 5 s intervals until the buffer clarified. Unbroken cells and cellular debris were removed by centrifugation at 5000 g for 20 min. The supernatant was further centrifuged at 100,000 g for 1 h at 4°C. The pellet was resuspended in 2% (W/V) sodium lauroyl sarcosinate (Sigma, USA) and incubated at room temperature for 30 min before additional ultracentrifugation. The concentration of outer membrane proteins was determined using a Bradford assay (Bradford, 1976 (link)) (Pierce, USA).

Prontosan wound irrigation solution (B. Braun; Melsungen, Germany), poviargolum powder (FSUE “SCTB ‘Technolog’”; Saint Petersburg, Russia), Xydifon (commercial name of etidronic acid 20% aqueous solution; OJSC “MosChemPharm” named after N.A. Semashko; Moscow, Russia), and Dioxydin 1% aqueous solution (OJSC “MosChemPharm” named after N.A. Semashko; Moscow, Russia) were purchased from the market. CP grade powders of sodium lauroyl sarcosinate (Sigma-Aldrich, St. Louis, MO, USA) and cocamidopropyl betaine (Evonik Industries, Essen, Germany) were helpfully provided by our colleagues from the Institute of Macromolecular Compounds of RAS and CP grade sodium hypochlorite 10% aqueous solution by our colleagues from the Faculty of Dentistry and Medical Technology of the Saint Petersburg State University.

To allow for dye penetration, the virus-treated samples were immediately combined with 200 µM PMA (Biotium, Hayward, CA, USA) and incubated in the dark for 5 min at room temperature. Then, samples were photoactivated for 10 min on each side with 40 W LED light (Dynebio, Seongnam, Republic of Korea) at 460 nm wavelength at room temperature and distance was 3 mm from the samples [1 (link)]. A control group was included in the study that was not treated with PMA or exposed to halogen light to examine if the dye treatment interfered with viral identification. Sodium lauroyl sarcosinate (Sigma-Aldrich, St. Louis, MO, USA) was used at 1.0% (w/v) to optimize the sarkosyl concentration and to examine its effect on HuNoV [18 (link)]. Sarkosyl solution was added to the PMA mixture at the same time as detergent treatment. PMA + sarkosyl-treated samples were incubated for 10 min at room temperature in the dark. An untreated control was included and unexposed to LED light to examine the ability of PMA + sarkosyl treatment to interfere with HuNoV detection [9 (link),17 (link),18 (link)]. Finally, before RT-qPCR experiments for discriminative detection of possibly infectious and noninfectious HuNoV viral particles, viral samples were exposed to the optimized PMA and sarkosyl pretreatments.

Bacterial cells of MDR strain of A. baumannii Ab186 and MDR strain of E. coli EcMCR+ were grown in Luria-Bertani (LB) broth in the logarithmic phase, incubated with 2 and 16 mg/L of tamoxifen metabolites mixture, respectively, for 4 or 24 h, and lysed by sonication. OMPs were extracted with sodium lauroyl sarcosinate (Sigma, Spain) and recovered by ultracentrifugation, as described previously [29 (link)]. OMP profiles were determined via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) using 10% SDS gels and 6 μg protein of OMPs, followed by SimplyBlue SafeStain gel (Invitrogen, Spain).

Slide-mounted FFPE tissue sections were incubated in xylene (3 × 5 min), then absolute ethanol (2 × 1 min), 95% (v/v) ethanol (2 × 1 min), 70% ethanol (2 × 1 min), and Milli-Q water (1 min). After air-drying, 60 sections of deparaffinized tissue were harvested with a microtome blade into a 15 mL low-protein-adsorption centrifuge tube, and suspended by sonication in 7.2 mL of phase-transfer surfactant (PTS) (10 mM sodium deoxycholate (SDC), 10 mM sodium lauroyl sarcosinate (SLS) (Sigma-Aldrich St. Louis, MO), 0.1 M Tris–HCl, pH 9.0) buffer. The suspension was stored at 4 °C until use.