Multiskan spectrophotometer

The C. albicans cells were freshly revived by subculture on the SD agar plate. A loopful of inoculum was introduced into the SD broth and cells were grown for 16 h at 37°C before use. Approximately 2×10³ cells/mL were then inoculated into the freshly prepared 50 mL sterile SD medium. Different concentrations, equivalent to 2MIC, MIC, MIC/2, of test compounds were added separately into the conical flasks containing inoculated medium and incubated at 37°C and 160 rpm. Strain specific concentration of FLC was used as positive control viz 40 μg/mL for C. albicans ATCC 90028, 20 μg/mL for C. albicans D27 (FLC-susceptible) and 40 μg/mL for C. albicans D15.9 (FLC-resistant). At predetermined time periods (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 22, and 24 after incubation with agitation at 37°C), 1 mL aliquot of each sample was removed from the conical flask and growth was measured tubidometrically at 600 nm using Thermo Multiskan spectrophotometer. Optical density was recorded for each concentration against time (h).

In both cells, the pro-inflammatory cytokines, including PGE2, TNF-α, IL-6, and IL-1β, were determined in the culture media. The media were collected after 24 h treatment of garcinol and used to quantify the level of secretory cytokines using sandwich ELISA Kits (Sigma Aldrich, Merck KGaA, Darmstadt, Germany). Briefly, 100 µL of each sample and standard were added to the wells and further incubated for 2.5 h with gentle shaking at room temperature. Next, the solutions were removed, and each well was washed 4 times with 1X wash solution. Afterward, 100 µL of the corresponding antibody was added to each well and incubated for 1 h with gentle shaking at room temperature. Next, each well was washed, and 100 µL of streptavidin solution was added and incubated for 45 min with gentle shaking at room temperature. Next, the TMB substrate reagent was added and incubated for 30 min with gentle shaking at room temperature in the dark. After that, the stop solution was added, and the absorbance was measured at 450 nm using a Multiskan Spectrophotometer (Thermo Scientific).

Cell viabilities of THP-1 and RAW 264.7 cells were measured by using a 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay [33 (link),34 (link)]. Briefly, 1 × 10⁴ cells per well were plated into a 96-well microtiter plate using a complete culture medium and incubated for 24 h. After incubation, the culture medium was removed. The medium with garcinol in the presence or absence of 500 ng/mL LPS (Sigma Aldrich, Germany) was added and subsequently incubated for 24 h. After that, 5 mg/mL of MTT solution (Sigma, St. Loius, MO, USA) was added to each well and subsequently cultured at 37 °C for 3 h. After that, the supernatant was replaced with 100 μL DMSO. The absorbance at 490 nm was evaluated in a Multiskan Spectrophotometer (Thermo Scientific, Rockford, IL, USA).
Before assessment of the effects of garcinol, the safety doses were determined by treating the cells with garcinol ranging from 10 to 100 μM without LPS exposure. For determining the effects of garcinol, the co-incubation of LPS and garcinol (10, 20, and 30 µM) or 5 µM of dexamethasone (Sigma Aldrich, Darmstadt, Germany) as a positive control anti-inflammatory agent were performed. In addition, the half-maximal inhibitory concentration (IC₅₀) was evaluated as previously described [33 (link),34 (link)].

Cell proliferation was measured in a 96-well plate using the WST-1 assay. GC cells were transfected with 50 nM siUC.145 or siCT, and cell viability was subsequently analyzed using the Multiskan spectrophotometer (Thermo Fisher Scientific) at 450 nm at time points from 0 to 72 h. For rescue experiments, cells were transfected with pcDNA-UC.145 48 h after siRNA transfection, and cell proliferation was subsequently evaluated.

PARP activity was determined as PARylation levels in cell extracts on a histone coated 96-well plate. It was measured using the HT Colorimetric PARP/Apoptosis Assay (Trevigen) and experiments were executed according to manufacturer’s protocol. Absorbance was detected with a Multiskan spectrophotometer (Thermo Fisher).