Thiazolyl blue tetrazolium bromide

Minimum inhibitory concentrations (MICs) of LmEO were calculated based on the broth microdilution method in 96 microplates [16 (link)] against a panel of 8 microorganisms representing different species of different ecosystems. Ten microliters of cell suspension were added to each test well. MIC (%) values were assessed as the lowest LmEO concentration that inhibited the visible growth of each tested bacterium [5 (link)]. As an indicator of microorganism growth, 25 μL of thiazolyl blue tetrazolium bromide (0.5 mg/mL) (Sigma-Aldrich, Taufkirchen, Germany) was added to the wells and incubated for 30 min. The colourless tetrazolium salt acts as an electron acceptor and was reduced to a red-coloured formazan product by biologically-active organisms.
The minimum bactericidal concentration (MBC) was defined as the lowest concentration at which 99% of the bacteria were killed. It was determined by removing 10 μL from each well and inoculating in MH plates in strings. After 24 h of incubation at 37 °C, the number of surviving organisms was determined. Available standards benzyl alcohol, linalool, terpien-4-ol and globulol were also tested under identical conditions to compare their activities with those of the studies essential oil.

Zinc acetate (Zn(OAc)₂, 99.99%), polyvinylpyrrolidone (PVP, Mw = 10000), hydrogen peroxide (H₂O₂, 30 wt. % in H₂O), manganese(II) chloride (MnCl₂, 99%), zinc chloride (ZnCl₂, 99.999%), zinc oxide nanoparticles (ZnO NPs, 20 wt. % in H₂O), zinquin ethyl ester (95%), 3-(N-morpholino)propanesulfonic acid (MOPS, 99.5%), sodium acetate (99%), acetic acid (99.7%), 2',7'-dichlorofluorescin diacetate (DCFH-DA, 97%), thiazolyl blue tetrazolium bromide (MTT, 97.5%), and propidium iodide (PI, 94%) were purchased from Sigma-Aldrich. Apoptosis kit with annexin V-FITC and PI, hydrogen peroxide assay kit, and calcein-AM were obtained from Fisher Scientific.

Curcumin (CAS Number 458-37-7, Sigma-Aldrich, Purity: ≧99.5%), demethoxyCurcumin (CAS Number: 22608-11-3, Sigma-Aldrich, Purity: ≧98%), bisdemethoxyCurcumin (CAS Number: 33171-05-0, Sigma-Aldrich, Purity: ≧98%), Thiazolyl Blue Tetrazolium Bromide (MTT) were purchased from Sigma–Aldrich (CAS Number: 298-93-1). β-actin (Cat No. GTX109639), p-p38 (Cat No. GTX24822), p-Akt (Cat No. GTX128414), p-Erk (Cat No. GTX24819), p-smad2 (Cat No. GTX133475), p-smad3 (Cat No. GTX129841), cleaved PARP (Cat No. GTX132329), cleaved caspase 3 (Cat No. GTX86952) antibodies were purchased from GeneTex. Annexin V Alexa Fluor 488 Ready Flow Conjugate (Lot number 2081235) and 7-aminoactinomycin D (7-AAD, Catalog number: A1310) both were purchased from Thermo Fisher Scientific.

Cells were plated in 96-well plates in quintuplicate overnight and subsequently treated as indicated. Cell viability was determined via MTT assay with 0.5 mg/mL thiazolyl blue tetrazolium bromide (Sigma-Aldrich), as previously described [44 (link)]. For ID8 cells, cell number was determined by cell counting and trypan blue exclusion.

HepG2, Hep3B and HL7702 cells were seeded in 24-well cell culture plates with 1 × 10⁴ in 0.5 ml complete medium per well, and infected with multiplicity of infection (MOI) 50 by Ad/AFP-Casp-AFP-amiR and Ad/AFP respectively in triplicates. Cell viability was determined by MTT assay using thiazolyl blue tetrazolium bromide (Sigma, St. Louis, MO, USA; M2128). At designated time point post infection, the infected cells were incubated with MTT solution with a final concentration of 0.5 mg/ml for 4 h at 37°C, 5% CO₂ and saturated humidity. Medium was aspirated, 250 μl isoproponal was added per well to dissolve purple crystals and 100 μl of the dissolved solution was transferred into a 96-well plate for measurement of absorbance at 570 nm with a 690 nm reference by Molecular Device Spectra Max M4 Microplate Reader. Relative cell viability was calculated with the non-infected cells as controls. Experiment was repeated at least twice.