Ez cytox cell viability assay kit

To assess the cell viability, 3D4/31 MΦ (5×10³ cells/well in 100 μL of media) were seeded in a 96-well cell culture plate (SPL Life Sciences, Pocheon, Korea). The cells were incubated for 12 h and treated with vehicle (distilled water; DW) or ASF (1.2, 12, and 120 μg/mL) for 24 h. Phorbol 12-myristate 13-acetate (PMA, 2 nM; Sigma-Aldrich, St. Louis, MO, USA) was then added for activation of MΦ. The final concentration of 10% (v/v) WST-1 reagent (EZ Cytox Cell Viability Assay Kit; DoGenBio, Seoul, Korea) was administered, followed by incubation for 2 h. The cell viability (optical density [OD] at 450 nm) was measured on a microplate reader (Sunrise, Tecan, Männedorf, Switzerland) and normalized to the vehicle (without PMA). For the growth curve analysis, 3D4/31 MΦ (5×10⁴ cells) were seeded in a 60 mm cell culture dish (SPL Life Sciences) and incubated for 12 h. ASF (120 μg/mL) was added to the cells and further incubated either with or without 2 nM PMA, and vehicle (DW). The medium was replenished and cells were treated again every 24 h, for 3 days. Growth curve was assessed by subjecting the cells to staining with 0.4% (w/v) trypan blue dye (Thermo Fisher Scientific, USA) and counting the cell number on a hemocytometer, for 3 days at every 24 h interval.

HGF-1 cells were seeded in a 35 mm confocal dish (SPL) at a concentration of 2 × 10⁴ cells/mL and incubated for 24 h. After incubation, cells were washed with phosphate-buffered solution (PBS, Gibco), treated with 1 mL of prepared composite eluates, and incubated for another 24 h. The negative control (NC) was treated with DMEM and the positive control (PC) was treated with 1 mM H₂O₂ (Sigma-Aldrich).
For live/dead assay, a Live/dead Viability kit (Invitrogen, Waltham, MA, USA) was used and observed under an inverted fluorescence microscope (DS-Ri2, Nikon Corporation, Tokyo, Japan) and a confocal laser microscope (LSM 700, Carl Zeiss, Thornwood, NY, USA). Live cells were observed with green fluorescence, and dead cells were observed with bright red fluorescence.
For WST-1 assay, the EZ-Cytox cell viability assay kit (DoGen Bio, Seoul, Korea) was used to determine cytotoxicity after 24 h of treatment. The optical density (OD) was determined at 450 nm using a microplate reader (AMR-100, Allsheng, Hangzhou, Zhejiang, China). Relative cell viability was calculated as the ratio of OD of experimental groups (TN, CX, and DN) to that of NC. The experiments were triplicated (n = 9).

Fetal human dermal fibroblasts (HDFs) were purchased from Genlantis (USA). The cells were maintained at 37 °C in a Dulbecco’s modified Eagle medium (DMEM; Welgene, Gyeongsan-si, Gyeongsangbuk-do, South Korea) containing 10% fetal bovine serum (FBS; Biological Industries, Beit Haemek, Northern districts, Israel) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA). The culture medium was changed every two days, and cells were passaged at subconfluency. Briefly, 10⁴ HDF cells were plated in each well of 96-well culture plates and stabilized for 24 h [24 (link)]. Consequently, the culture medium was replaced with 1 mL of DMEM with various concentrations of three different types of microspheres (1:1 (L), 1:2 (M), 1:3 (S)). After incubation for 24 h, cell viability was measured by an EZ-Cytox cell viability assay kit (DoGenBio, Seoul, South Korea) [25 (link)]. The cells were washed with phosphate-buffered saline (PBS) and then treated with 10 μL of the EX-Cytox solution and 100 μL of DMEM at 37 °C for 3 h according to the manufacturer’s instructions. After incubation, the absorbance was measured at 450 nm using a microplate spectrophotometer (SpectraMax Plus, Molecular Devices, San Jose, CA, USA). The absorbance values were normalized based on that of the negative control cell group (without microspheres) (n = 3).

The cytotoxicity of the extracts on the melan-a cells was measured using the EZ-CyTox Cell Viability Assay Kit (DoGenBio, Seoul, Republic of Korea). The cells were seeded at 2 × 10⁴ cells/well in a 96-well plate and incubated at 37 °C for 24 h. The culture medium was replaced with the varying concentrations of extracts, and the plate was further incubated for 72 h. The working solution of EZ-CyTox (10-fold dilution in 1× PBS; 110 µL/well) was applied to the cells, and the plate was incubated at 37 °C for 4 h. After incubation, 100 µL of solution from each well was transferred to a new 96-well plate, and the absorbance at 450 nm was recorded using a SpectraMax^® ABS Plus Microplate Reader (Molecular Device, San Jose, CA, USA). Culture medium (alone)-treated cells were used as the control group, and the cell viability was assessed using the following formula: $$\mathrm{Melan-a} \mathrm{cell} \mathrm{viability} \left(\%\right)=\frac{\mathrm{Absorbance} \mathrm{at} 450 \mathrm{of} \mathrm{treatment}}{\mathrm{Absorbance} \mathrm{at} 450 \mathrm{of} \mathrm{control}} \times 100.$$