Cytotoxicity evaluation of the extracts for mouse fibroblasts line L929 by MTT method

Mouse fibroblasts L929 (ATTC, CCL-1) were cultured as monolayers in 25 cm² tissue culture flasks (BD Falcon) in RPMI 1640 medium (Sigma/Merck, Germany) supplemented with 10% fetal bovine serum (Biological Industries) and 100 U/mL penicillin, 100 μg/mL streptomycin, 0,292 mg/mL L-glutamine (Gibco/Intvitrogen). Every three days the monolayer was treated with trypsin/ethylene diamine tetraacetic acid (BioWest) and sub-cultured. Cells were maintained at 37°C under a humidified atmosphere containing 5% CO₂. Cytotoxicity was assessed using the colorimetric MTT assay [57]. Briefly, confluent monolayer was trypsinized, the suspension of 1 × 10⁵ cells/mL prepared, 100 μL per well of 96-well tissue culture plate (Greiner bio-one) seeded, and incubated at 37°C in a humidified 5% CO₂ atmosphere. For background absorption, some wells were remained cell-free (blank control). After 24h the growth medium was discarded, and 100 μL of extract diluted in RPMI 1640 medium (supplemented as earlier) was added at the following concentrations: 1000, 500, 250, 125, 62.5, 31.2, 15.6, and 7.8 μg/mL (n = 3). In each case the concentration of extracts’ solvent (Et-OH) did not exceed 2,5% and did not disturb fibroblast growth. Three wells were remained untreated as negative control. Following 24 h incubation under the conditions as earlier, the treatment medium were removed and 50 μL/well of MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma/Merck) in phosphate-buffered saline (1 mg/mL) after filtration through a 0.2 μm syringe filter was added and incubated for 2 h as above. The MTT solution was removed and replaced with 150 μL/well of DMSO (dimethyl sulfoxide; Sigma/Merck) to dissolve the blue formazan product formed by living cells. Additionally, 25 μL/well of Sörensen’s glycine buffer (glycine 0.1 M, NaCl 0.1 M, pH:10.5 with 0.1 NaOH) was added. The absorbance (A) was detected at 570 nm with the use of the microplate reader Multiskan EX (Thermo Labsystem-Fischer, USA). The viability of cells cultured in full RPMI 1640 alone (negative control) was considered as 100%, and the percentage of cell viability was calculated using the formula below:

Survival rate (%) = [(As–Ab)/(Ac–Ab)] × 100

As–absorbance of test sample (the cells exposed to the extract)

Ac–absorbance of negative control (the cells in medium alone)

Ab–absorbance of blank control

All raw data is added as S2 Raw data.