3 3 dipropylthiadicarbocyanine iodide disc3 5

The cytoplasmic membrane depolarisation activity of HICA was determined using the voltage sensitive fluorescence dye 3,3′-Dipropylthiadicarbocyanine iodide [DiSC₃(5)] (Sigma-Aldrich, Czech Republic) as described previously [22 (link)] with minor modifications. B. cereus NZRM5, B. cereus M4, S. aureus NZRM917, E. coli O157:H7 NCTC 1200, E. coli AGR3789, P. aeruginosa NZRM981, and P. aeruginosa NZRM4034 were grown overnight in MHB at 35°C and diluted to 0.3 OD₅₉₅ with fresh MHB supplemented with 0.5 mg/mL Bovine Serum Albumin (BSA) (Sigma-Aldrich, USA). The diluted bacterial cell suspension (130 μL) was transferred to an opaque-walled 96 well plate and combined with 20 μL of 60 μM DiSC₃(5) (Sigma-Aldrich, Czech Republic). The fluorescence quenching was then measured using the Varioskan™ LUX multimode microplate reader at an excitation wavelength of 610 nm and emission wavelength of 660 nm, until a stable fluorescence signal was achieved. Following the addition of HICA (4 mg/mL) or sterile water to corresponding wells, fluorescence was immediately measured and monitored for 25 min at 60 s intervals with 10 s of vigorous shaking before each measurement. Samples with added sterile water served as the untreated controls and used to compare the change in the fluorescence intensity of HICA-treated group.

Novozym 435 was obtained from Novozymes (Copenhagen, Denmark). Lipase PS-C was purchased from Sigma-Aldrich (Milwaukee, WI, USA). Sodium metal (large pieces in kerosene) was purchased from Fisher Scientific (Pittsburgh, PA, USA). 3,3′-Dipropylthiadicarbocyanine iodide (diS-C3(5)) and polymyxin B were purchased from Sigma-Aldrich. Tetrahydrofuran (THF), chloroform (CHCl₃), methanol (CH₃OH) and dimethyl sulfoxide (DMSO) were purchased from Fisher Scientific (Pittsburgh, PA, USA). All bacteria culture media and agars were purchased from Sigma-Aldrich (Milwaukee, WI, USA). All other chemicals and solvents were purchased in the highest available purity from Sigma-Aldrich and were used without further purification.

Thiazolyl blue tetrazolium bromide (MTT), N-phenyl-1-naphthylamine (NPN), 3,3′-dipropylthiadicarbocyanine iodide (DiSC₃(5)), SYTOX green, propidium iodide (PI), dimethyl sulfoxide (DMSO), cefotaxime, colistin, and ampicillin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vancomycin was purchased from LPS Solution (Deajeon, Korea). Sheep red blood cells (sRBCs) were purchased from MB Cell (Seoul, Korea).

3,3′-Dipropylthiadicarbocyanine iodide (DiSC₃(5)), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Triton X-100, DMSO, glutaraldehyde solution, and lipopolysaccharides (LPSs) from E. coli O111:B4 were purchased from Sigma-Aldrich (Burlington, USA). DAPI, FM 4-64 dye, SYTOX green nucleic acid stain, SYTO 9 green fluorescent nucleic acid stain, phosphate-buffered saline (PBS) tablets, and ProLong glass antifade mountant were purchased from Thermo Fisher (Waltham, USA). The purified albofungin was further conjugated with FITC by a TGpeptide company (Jiangsu, China). Vibrio alginolyticus 1597 and V. parahaemolyticus 1482 strains were given by Prof. Chen Sheng from the City University of Hong Kong.

Staphylococcus aureus (ATCC 29213) cells were cultured overnight in TSB, then washed once and resuspended at an OD_600nm of 0.1 in assay buffer containing 5 mM HEPES, 5 mM glucose and 0.1 M KCl, at pH 7.4. Bacteria were dispensed into a 96-well black plate and 3,3′-dipropylthiadicarbocyanine iodide [diSC3(5)] (Sigma-Aldrich Co., LLC, MO, United States) was added to a final concentration of 1 μg/mL and incubated with the cells for 30 min. Test peptide was then added to the final concentration 1-50 μg/mL, the mixture incubated for 30 min and fluorescence measured using a Cytation 5 microplate reader (BioTek) with an excitation wavelength of 622 nm and emission at 670 nm.

3,3′-Dipropylthiadicarbocyanine iodide (DiSC₃(5)), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Triton X-100, DMSO, glutaraldehyde solution, and lipopolysaccharides (LPSs) from E. coli O111:B4 were purchased from Sigma-Aldrich (Burlington, USA). DAPI, FM 4-64 dye, SYTOX green nucleic acid stain, SYTO 9 green fluorescent nucleic acid stain, phosphate-buffered saline (PBS) tablets, and ProLong glass antifade mountant were purchased from Thermo Fisher (Waltham, USA). The purified albofungin was further conjugated with FITC by a TGpeptide company (Jiangsu, China). Vibrio alginolyticus 1597 and V. parahaemolyticus 1482 strains were given by Prof. Chen Sheng from the City University of Hong Kong.

SDS, TFE, thiazolyl blue tetrazolium bromide (MTT), NPN, 3,3′-dipropylthiadicarbocyanine iodide (DisC₃-5), SYTOX green, propidium iodide (PI), dimethyl sulfoxide, meropenem and polymyxin B were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vancomycin was purchased from LPS solution (Daejeon, South Korea).