Round bottomed 96 well microplates

PBMC were incubated in round-bottomed 96-well microplates (Corning Incorporated) at 1 × 10⁵ cells/well in complete RPMI 1640 medium. After 24 h of incubation, cells were treated with methanol extracts at concentrations ranging from 100 µg/mL to 500 µg/mL, using 5 µg/mL concanavalin A (Con A) as a positive control and untreated culture medium as a negative control. Cells were incubated for 48 h at 37 °C in an atmosphere of 5% CO₂ in air, and cell viability was determined using the colorimetric MTT reduction assay, as explained above [18 (link)]. Results were expressed as the proliferation index, which was calculated using the following Formula (4) [21 (link)]: $$\mathrm{P}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x}=\frac{{\mathrm{O}\mathrm{D}}_{570} \mathrm{T}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}-{\mathrm{O}\mathrm{D}}_{570} \mathrm{N}\mathrm{e}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}{{\mathrm{O}\mathrm{D}}_{570} \mathrm{N}\mathrm{e}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}$$
In addition, we determined the half maximal effective concentration (EC₅₀) of plant extracts for PBMC proliferation. The percentage of proliferation was calculated by multiplying the proliferation index by 100 [22 (link)].

Cells were incubated in round-bottomed 96-well microplates (Corning Incorporated, Corning, NY, USA) at concentrations of 1 × 10⁴ L5178Y-R cells/well and 1 × 10⁵ PBMC/well in complete RPMI 1640 medium. After 24 h of incubation, cells were treated with methanol extracts at concentrations ranging from 3.9 µg/mL to 1000 µg/mL. The antineoplastic vincristine sulfate (VC) (Hospira, Warwickshire, UK) at 100 µg/mL was used as a positive control and untreated culture medium was used as a negative control [15 (link)]. Cells were incubated for 48 h at 37 °C in an atmosphere of 5% CO₂ in air and cell viability was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT; Affymetrix, Cleveland, OH, USA) colorimetric method by adding 15 µL of MTT/well (0.5 mg/mL final concentration) and incubating the plate at 37 °C for 3 h [18 (link)]. Plates were then decanted, formazan crystals were dissolved with 100 µL of DMSO, and optical densities (OD) were measured at 570 nm in a microplate reader (MULTISKAN GO; Thermo Fisher Scientific, Waltham, MA, USA). Percentage growth inhibition was calculated as follows (2): $$\% \mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{w}\mathrm{t}\mathrm{h} \mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=100-\left(\frac{{\mathrm{O}\mathrm{D}}_{570} \mathrm{T}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}{{\mathrm{O}\mathrm{D}}_{570} \mathrm{U}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}\times 100\right)$$