Methyl thiazolyl tetrazolium (mtt)

The MTT assay was used to determine cell viability. When
bleomycin enters a cell, it causes cell death. Therefore, the
cell viability assay can indicate cell permeability. The MTT
(Invitrogen, Gibco, USA) assay was conducted as follows:
20 μl MTT solution [5 mg MTT/ml in phosphate-buffered
solution (PBS)] was added to wells and after a four hour
incubation period, dimethyl sulfoxide (DMSO) was added
to the wells and the solution was mixed. Optical density was
read by an optical Elisa Reader (BioTek,Cytation 5, USA) at
a 570 nm wavelength filter (21 ).

HL-60 cells (2 × 10⁴ cells) were plated into 96-well culture plates and treated in triplicate with or without the compounds identified in the CD assays. The compounds were added from stock solutions in DMSO and the final concentration of DMSO in the medium was 0.2%. After 72 h, 20 μL 5 mg/ml MTT (Molecular Probes, Life Technologies Corporation) was added to each well and incubated for 3 h. After the incubation, medium containing drug and MTT was removed, 200 μL DMSO was added to the cells, and the plate was shaken for 5 min. The number of viable cells was assessed by spectrophotometry at 570 nm using a BioTek Synergy4 microplate reader and calculated as the percentage of absorbance of treated cells relative to that of solvent controls. Results were expressed as a percentage of inhibition and the EC₅₀ was calculated using the Hill equation.

To determine the effect of CM from T-DPSC (T-DPSC-CM) on proliferation of endothelial cells, HUVECs were seeded on a 96-well plate at a density of 3000 cells per well. After 24 h, the mixtures of DPSC-CM or T-DPSC-CM with EGM2 (1:1) were added to the corresponding wells. Cell numbers were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Molecular Probe, OR, USA) at different time points of 0, 24, 48, and 72 h. Briefly, the medium was replaced with a 100-μL fresh medium and 10 μL 12 mM MTT solution was added to each well. After incubation for 4 h at 37 °C, the medium was removed leaving only 25 μL in the well. Then, 50 μL DMSO was added to each well and mixed thoroughly. After incubation for 10 min at 37 °C, the absorbance of each well at 540 nm was measured with a SpectraMax®M2microplate reader (Molecular Devices, CA, USA).

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): $$\% \mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{w}\mathrm{t}\mathrm{h} \mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=100-\left(\frac{{\mathrm{O}\mathrm{D}}_{570} \mathrm{T}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}{{\mathrm{O}\mathrm{D}}_{570} \mathrm{U}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}\times 100\right)$$

L5178Y-R, HT-29, MCF-7, and MA-104 cell suspensions were cultured at a density of 1 × 10⁴ cells/well and PBMCs at 1 × 10⁵ cells/well into flat-bottomed 96-well plates (Corning Incorporated, Corning, NY, USA) in complete culture medium. After 24 h of incubation, cells were treated with 31 µg/mL, 62.5 µg/mL, 125 µg/mL, and 250 µg/mL of endophytic fungus methanol extracts for 48 h at 37 °C in 5% CO₂. Tumor cell growth was then evaluated using the colorimetric 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT; Affymetrix, Cleveland, OH, USA) reduction assay by adding 15 µL of MTT (0.5 mg/mL final concentration) and incubating at 37 °C for an additional 4 h. Formazan crystals were dissolved with DMSO, and optical densities (OD) were measured at 570 nm in a MULTISKAN GO microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Cell growth inhibition percentage was calculated as follows: % Growth inhibition = 100 − [(OD₅₇₀ in extract-treated cells/OD₅₇₀ in untreated cells) (100)], using 0.05 µg/mL vincristine sulphate (VC; Hospira, Warwickshire, UK) as a positive control. Logarithmic scale concentrations were plotted against % cytotoxicity to determine IC₅₀ values, which were used to determine the selectivity index (SI). This index was calculated by dividing the IC₅₀ of normal cells by that of tumor cells [20 (link)].