Flat bottomed 96 well plate

Extracts from GP were filtered through a 0.22 μm syringe filter and prepared in sterile deionized water at a concentration (S/L ratio) of 0.25 mg/mL, 1.00 mg/mL, 1.75 mg/mL, and 2.5 mg/mL.
The antiproliferative effect of the extracts of GP was determined by cell viability using the MTT assay [30 (link)]. Cells were seeded in 96-well flat-bottomed plates (Greiner, Frickenhausen, Austria) at a concentration of 2 × 10⁴ cells/mL and left overnight in the CO₂ incubator to attach to the plate surface. 72 hours (72 h) after addition of the extracts from GP, the growth medium was discarded and 5 mg/mL MTT was added. After 4 hours of incubation at 37 °C, the water-insoluble MTT formazan crystals were dissolved in DMSO. The absorbance was measured at 595 nm using an Elisa microplate reader (iMark, BIO RAD, Hercules, CA, USA). Control cells were grown under the same conditions. Blank means medium without cells containing MTT. All experiments were performed at least three times in triplicate. The percentage of cell viability (CV, %) was calculated according to eq. (1): $$\mathsf{CV}=\frac{A_{\mathit{sample}}-A_{\mathit{background}}}{A_{\mathit{control}}-A_{\mathit{background}}}\bullet 100\left(\%\right)$$
GI₅₀ value, defined as compound concentration (mg/mL) that results in a 50% reduction in cell viability, was calculated and used as a parameter for comparing the inhibitory effect between GP samples.

Affinity capture (GST pulldown) experiments were performed in 96-well flat-bottomed plates (Greiner) using magnetic glutathione beads (Thermo Scientific). For capture of prey proteins produced by cell-free expression in wheat germ lysate, 0.5 nmol of purified GST-tagged bait protein was incubated with magnetic glutathione beads at 4°C for 10–20 min. The beads were washed three times with wash buffer (20 mM Tris, 200 mM NaCl, 0.1% NP-40, 1 mM DTT, and 1 mM EDTA), incubated with wheat germ cell-free expression reactions at 4°C for 60 min, and then washed four times in wash buffer. Bound protein was eluted using wash buffer supplemented with 50 mM reduced glutathione and then boiled in SDS–PAGE loading buffer for SDS–PAGE and immunoblotting. For capture of purified prey proteins expressed in E. coli, 1.5 nmol of GST-tagged bait protein was incubated with magnetic glutathione beads for 15 min at 4°C and then washed three times with 20 mM Tris, 200 mM NaCl, 0.1% NP-40, and 1 mM DTT. The bait-loaded beads were incubated with 1.5 nmol of purified prey protein at 4°C for 60 min, washed four times, and bound protein was eluted using wash buffer supplemented with 50 mM reduced glutathione before boiling in SDS–PAGE loading buffer for SDS–PAGE analysis.

The cytotoxicity of the crude MeOH leaf extract of 11 C. nutans samples was measured using the Cell Counting Kit-8 (CCK-8) (Sigma-Aldrich, USA) according to the manufacturer’s instructions. Briefly, the D24 and NHDF cells were seeded at 5 × 10³ cells/well in 96-well, flat bottomed plates (Greiner, Austria). After 24 h, the cells were treated with the different extracts at 2 mg/mL prepared in DMSO (Sigma-Aldrich) with a final concentration of < 0.1 % (v/v) and incubated for 72 h. Cytotoxicity was measured at 450 nm using a microplate reader (CLARIOstar, BMG Labtech, Germany). The percentage of viable cells was determined relative to the vehicle control (<0.1 % DMSO) using the following equation: $\mathit{Viable}\mathit{cell}\left(\%\right)=\left(\mathit{absorbance}\mathit{of}\mathit{sample}/\mathit{absorbance}\mathit{of}\mathit{control}\right)\times 100$
The vehicle control was expressed as 100 %. Only the most active C. nutans sample (CT5) against the D24 cells as determined in this assay were used for further experiments.