Human total mmp 1 duoset elisa kit

HaCaT cells were seeded at 2 × 10⁵ cells/well in 12-well plates. Then, 24 h later, they were pretreated with TCQA (15) (5, 10, or 20 μM) for 4 h, washed with PBS, and then exposed to UVB (15 mJ/cm²) and TCQA (15) overnight in 1% FBS medium. After exposure to UVB, culture supernatants were collected and centrifuged at 13,000 rpm for 5 min. Concentrations of matrix metalloproteinase-1 secreted into culture media were determined using human total MMP-1 enzyme-linked immunosorbent assay (ELISA) kits (Human Total MMP-1 DuoSet ELISA kit; R&D Systems, Minneapolis, MN, USA). Absorbances at 450 nm were read using a microplate reader (Tecan Infinite M1000 Microplate Reader, Tecan).

HDFs were seeded into 48-well plates, and the density of the cells was 2 × 10⁴ cells/200 μL in each well. After this step, the cells were incubated in a cell incubator for 24 h prior to the ELISA assay. Then, cells were arranged in the same cycle by changing the old medium to a fresh medium without FBS. After 24 h, WAD was treated on the HDFs for 1 h and immediately exposed to TNF-α at 20 ng/mL. After 12 h, the supernatant was collected from the pretreated cells for the determination of the secretion of IL-6 and IL-8. Meanwhile, to measure the secretion of MMP-1 and procollagen I α1 (COLIA1), the supernatant was collected from the cells after 24 h. Secretion proteins were evaluated using a Human IL-6 Quantikine ELISA Kit (CAT No. D6050, R & D systems, Minneapolis, MN, USA), a Human IL-8/CXCL8 DuoSet ELISA (CAT No. DY208), a Human Total MMP-1 DuoSet ELISA kit (CAT No. DY901, R & D systems), and a Human Pro-Collagen I alpha 1 DuoSet ELISA (CAT No. DY6620-05, R & D systems). The optical density for protein secretion was measured using a SPARK 10M spectrophotometer (Tecan Group Ltd., Männedorf, Switzerland) with the wavelength set at 450 nm. The protein secretion results were calculated using a standard curve and are presented as fold-change values.

Iscove’s modified Dulbecco’s medium (IMDM), Dulbecco’s Modified Eagles medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Life Technologies Inc. (Grand Island, NY, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan). N-Succinyl-Ala-Ala-Ala-p-nitroanilide (STANA), elastase from porcine pancreas, all-trans retinol, and adenosine were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Procollagen Type I C-peptide (PIP) EIA kit was purchased from Takara Bio Inc. (Kusatsu, Shiga, Japan). Human Total MMP-1 DuoSet ELISA kit was obtained from R&D Systems (Minneapolis, MN, USA).