3 4 5 dimetylthiazol 2 yl 2 5 diphenyltetrazolium bromide

Lipopolysaccharide (LPS; from Pseudomonas aeruginosa), Lipoteichoic acid (LTA; from Staphylococcus aureus), 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), propidium iodide (PI), n-phenyl-1-naphthylamine (NPN), HEPES, and 3,3′-dipropylthiadicarbocyanine iodide (DisC₃-5) were obtained from Sigma-Aldrich (St Louis, MO, USA).
Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 25923 were obtained from the ATCC (American Type Culture Collection, Manassas, VA, USA), and Acinetobacter baumannii KCTC 2508 and Bacillus subtilis KCTC 2217 were obtained from the KCTC (Korean Collection for Type Cultures, Jeongeup-si, Jeollabuk-do, Korea). Human skin epithelial cells (HaCaT cells) were obtained from ATCC.

The cells were plated in 96-well plates at a density of 2 × 10⁴ cells per well. After incubating overnight, the cells were treated with HPA3, HPA3P, HPA3P2, oxaliplatin, and 5-FU at various concentrations. After the cells had incubated for 6 h or 24 h, the culture medium was replaced with fresh medium. Twenty microlitres of a 5 mg/ml stock solution of 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma Aldrich) was added to each well, and the cells were incubated for 1 h at 37°C in a humidified atmosphere of 5% CO₂. After removal of the supernatant, 100 μl of DMSO was added to each well. The plates were subsequently incubated on a horizontal shaker for 20 min at room temperature, and the optical density was measured at an absorbance of 570 nm using a microplate reader (SPECTRA MAX PLUS, Molecular Devices, CA, USA).