Medium 106

HaCaT keratinocyte cells, acquired from the American Type Culture Collection (ATCC, VA, USA), were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin in 5% CO₂ under a humidified atmosphere at 37 ℃. Human dermal fibroblast (HDF) cells were obtained from Gibco (CA, USA). The cells were cultured in Medium 106 (Gibco) supplemented with 1 × Low Serum Growth Supplement (LSGS, Gibco) in 5% CO₂ under a humidified atmosphere at 37 ℃. Cell viability was measure by using an EZ-Cytox Cell viability assay kit (Dail Lab, Korea). HaCaT cells were seeded at 1 × 10⁴ cells/well in 96-well plates and treated with Ag₂S NPs and Li-doped Ag₂S NPs at various concentrations for 24 and 48 h. HDF cells were seeded at 3 × 10³ cells/well in 96-well plates and treated with Ag₂S NPs and Li-doped Ag₂S NPs at various concentrations for 24 h. The WST reagent solution (10 μL) was added to each well of a 96-well microplate that contained 100 μL of cells. The plate was then incubated for 4 h at 37 °C. HDF cells from passage number 10 were used for the study. The absorbance was measured at 450 nm using a microplate reader.

HNDFs (Gibco BRL, USA; isolated from the skin of newborn male) were plated at a density of 2.5 x 10³ cells/cm² and cultured in Medium 106 (Gibco BRL) supplemented with low serum growth supplement (Gibco BRL) at 37 °C in humid air with 5 %(v/v) CO₂. The medium was replaced every other day until the culture is approximately 80 % confluent, and after that, exchanged every day. HL-1 cells (EMD Millipore, USA), murine cardiomyocyte cell line, were cultured in Claycomb medium (Sigma Aldrich) containing 10 %(v/v) fetal bovine serum (FBS; Gibco BRL), 0.1 mM norepinephrine (Sigma Aldrich), 2 mM L-glutamine (Gibco BRL), and 100 units/ml penicillin, and 100 μg/ml streptomycin (Gibco BRL). HL-1 cells were maintained at a high cell density to prevent dedifferentiate, so they were passaged at 1:3 split ratio only at 100% confluency. The culture plates for HL-1 cells were coated with 0.02 %(w/v) gelatin (Sigma Aldrich) and 5 μg/mL fibronectin (Sigma Aldrich).

Peripheral blood mononuclear cells were isolated from the buffy coat of healthy adult donors (National University of Singapore Blood Donation Centre). Ficoll-Paque PREMIUM (GE Healthcare) gradient centrifugation was performed according to the manufacturer's instructions. Briefly, the mononuclear cell layer was isolated and washed with PBS supplemented with 2% FBS (GE healthcare) and 1 mM EDTA, to remove platelets. Monocytes were subsequently purified by negative selection using the Human Monocyte Enrichment Kit (StemCell Technologies) according to the manufacturer's instructions.
Primary monocytes and U937 cell line were cultured in RPMI (Life Technologies) while A549 cells were cultured in DMEM (Life Technologies) supplemented with 10% FBS and 1% (v/v) penicillin and streptomycin (Life Technologies). The cells were grown at 37°C and 5% CO₂. For differentiation of U937 into monocytes, 5 × 10⁵ cells/mL of pre-differentiated cells were induced with 30 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 24 h, before changing to fresh media. The cells were cultured for another 48 h to allow for full differentiation and thereafter used for downstream assays.
The primary neonatal human fibroblasts (Life Technologies) were routinely grown in medium 106 (Life Technologies) before RNA extraction.

HDFα cells were purchased from Life technologies (cat. No. C0135C) and cultured in a specific pathogen-free cell culture laboratory at 37°C in 5% CO2. HDFα cells were maintained in Medium 106 (Life Technologies Corporation, Carlsbad, USA) supplemented with LSGS (Life Technologies Corporation, Carlsbad, USA) and amphotericin B (Sigma). 160 μg/ml gentamycin (Sandoz, Sandoz GmbH, Austria).

The screening experiments were conducted in normal primary HDFs that were isolated from the skin of a young adult (Thermo Fisher Scientific, Waltham, MA, USA). The cells were maintained in Medium 106 that was supplemented with low serum growth supplement (LSGS) and 1% antibiotic–antimycotic (all reagents for cell culture were purchased from Thermo Fisher Scientific) at 37 °C and 5% CO₂ in a humidified atmosphere and sub-cultured per the manufacturer’s instructions. The experiments were conducted with cell passages 6–8. Cell viability and confluence were determined in five replicates per treatment, and ELISA was performed with three replicates per treatment.

Human dermal fibroblast neonatal cell line (HDF-N) derived from neonatal foreskin (C-004-5C, Thermo Fisher Scientific, Carlsbad, CA, USA), and human dermal fibroblast adult cell line (HDF-A) isolated from adult skin (C-013-5C, Thermo Fisher Scientific, Carlsbad, CA, USA) were cultured in a humidified atmosphere under the standard conditions (37 °C, 5% CO₂) in Medium 106 (Thermo Fisher Scientific, Carlsbad, CA, USA) supplemented with Low Serum Growth Supplement (Thermo Fisher Scientific, Carlsbad, CA, USA), according to manufacturer’s instruction. The number of passages of cells that were used in analyses were 11 and 14 for (HDF-N) and 14 (HDF-A,) respectively.