Imt 2

The cytotoxicity of the extracts was determined using the 3-(4,5-dimethythiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as follows [36 (link)]: 1.0 × 10⁴ human fibroblast cells/well (CCD-986sk, ATCC, Manassas, VA, USA) were inoculated into a 96-well plate and further incubated in a CO₂ incubator at 37°C for 6 hours. Subsequently, 200 μL of the three extracts (GE, WE, and FE) with various concentrations was added to the cells, and the cells were cultured for 24 hours. MTT solution (5 μg/mL) was added to each well, and the supernatant was removed 4 hours later. Thereafter, 10 μL of acid-isopropanol (0.04 N HCl in isopropanol) was added to the solution, and the absorbance of the solution was measured using a microplate reader at a wavelength of 565 nm. The cell cytotoxicity was expressed as the percent ratio of the cell number in the sample treated with a certain concentration to the cell number in the untreated control sample [37 (link)].
The morphology and density of the fibroblast cells treated with the various extracts at doses of 0.01% and 0.5% were also observed under an inverted microscope (IMT-2, Olympus, Tokyo, Japan) and compared with those of the control (untreated cells).

Titers of virus produced in HaCaT wild type or COSMC-/- keratinocytes were determined on Green monkey kidney (GMK) cells. Cell monolayers were infected with serial dilutions of virus and allowed to attach. After 1 h the inoculum was removed and the cells overlaid with medium containing 1.5% methylcellulose (Sigma-Aldrich), 2.5% FCS, 100 IU/mL penicillin and 100 μg/mL streptomycin (in HBSS (Sigma-Aldrich) + DMEM (Gibco, Life Technologies) at a ratio of 1:1). After 48 h incubation, the overlay medium was removed, the cells fixed with 1% crystal violet (in 70% EtOH:37% formaldehyde:acetic acid 20:2:1), washed three times with water and allowed to dry. The resulting plaques were inspected and counted using a light microscope (Olympus IMT-2).

HK-2 cells in different groups were washed three times with PBS and viewed under an inverted microscope (Olympus IMT-2, Tokyo, Japan). The morphology of the cells was observed under 100× magnification.

Adherent amnion derived stromal cells in passage 0 (P0) were grown to 80% confluence and the monolayers rinsed with PBS. Accutase (Gibco, Thermo Fisher Scientific) was added, incubated at 37°C until cells detach and stopped by adding 5 ml of PBS. The cells were centrifuged at 300 g for 5 min and counted. Five thousand cells/25 μl drop were pipetted on a lid from a 100 mm petri dish (Greiner Bio-One, Kremsmünster, Austria) and incubated as hanging drops at 37°C in 5% CO₂ humidified environment. The aggregate formation was monitored by a stereo microscope (Olympus IMT2) and spheroids were collected after 2 days and forwarded to characterization by flow cytometry.