Human pro collagen 1 alpha 1 duoset elisa

Heat-inactivated fetal bovine serum (FBS, HyCylone™) and PBS (10X), pH 7.4 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Dulbecco’s modified Eagle’s medium high glucose 4.5 g/L (DMEM), penicillin-streptomycin solution, trypsin-EDTA (0.25%), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS substrate), 3,3′,5,5′-tetramethylbenzidine (TMB substrate), human glycated albumin, elastase from human leukocytes, N-succinyl-Ala-Ala-Ala-p-nitroanilide, oleanolic acid, hyaluronidase from bovine testes, 4 (dimethylamino)benzaldehyde (DMAB), hyaluronic acid sodium salt from cockscomb, sodium aurothiomalate hydrate (SATMH) and Collagenase Activity Colorimetric Assay Kit (MAK293) were purchased from Merck (Waltham, MA, USA). ELISA Human IL-6 Kit (900-K16) and ELISA Human IL-8 Kit (900-K18) were purchased from PeproTech (London, UK). Human Total MMP-1 DuoSet ELISA (DY901B-05), Human MMP-2 Duo-Set ELISA (DY902), and Human Pro-Collagen I alpha 1 DuoSet ELISA (DY6220-05) were purchased from R&D Systems (Minneapolis, MI, USA).

The concentrations of interleukin 6 (IL-6), interleukin-8 (IL-8/CXCL8), and pro-collagen Iα were measured using Interleukin-6-ELISA-BEST (A8768, Vector-Best, Novosibirsk, Russia), Interleukin-8-ELISA-BEST (A8762, Vector-Best, Russia), and Human Pro-Collagen I alpha 1 DuoSet ELISA (DY6220-05, R&D Systems, Minneapolis, MN, USA) kits, respectively. The assays were carefully performed, according to the manufacturer’s protocols.

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.

Immunofluorescence (Anti-Collagen I, Abcam, UK) and enzyme-linked immunosorbent assay (ELISA) (Human Pro-Collagen I alpha 1 DuoSet ELISA, R&D systems, USA) were used to determine the type I collagen in HFF-1 cells.

ELISA was performed as described previously^{43 (link)}. 0.3 × 10⁴ corneal fibroblasts were seeded into 96-well plates in DMEM/F-12 supplemented with10% FBS for 24 h before treatment. Fibrosis was induced as described earlier. Desired wells were pretreated with either 10 μM mitoTEMPO or 20 nM TPCA. Supernatants were collected 2 days after treatments. Secretion of IL-8 was assessed with Human CXCL/IL-8 DuoSet (R&D Systems, #DY208), pro-collagen I was assessed with Human Pro-Collagen I alpha 1 DuoSet ELISA (R&D Systems, #DY6220), collagen III with Human Collagen, type III, alpha 1 (COL3A1) ELISA kit (Cusabio, Houston, TX, USA, # CSB-E13446h) and collagen V with Human Collagen, type V, alpha 1 (COL5A1) ELISA kit (Cusabio, # CSB-E13447h) according to the manufacturer protocol.

Type I collagen was measured in cell culture supernatants using Human Procollagen I alpha 1 DuoSet ELISA (R&D Systems) and IL-11 was measured in cell culture supernatants using IL-11 Human ELISA kit (Thermo Fisher Scientific) according to the manufacturer’s protocol.

We seeded NHDFs at 2 × 10⁴ cells/well in 48-well plates, incubated them for 24 h, and then replaced the medium with a serum-free medium to create starvation conditions. After 24 h, the NHDF was treated with 3,5,7-trimethoxyflavone for 1 h, followed by exposure to 20 ng/mL of TNF-α. To measure protein expression for IL-1β, IL-6, and IL-8, the medium was collected from cells after 12 h. To measure protein expression for MMP-1 and procollagen I α1 (COLIA1), the medium was collected from cells after 24 h. Protein secretion was determined using a human Total MMP-1 DuoSet ELISA kit and a Human Pro-Collagen I alpha 1 DuoSet ELISA (R&D systems, Minneapolis, MN, USA). The absorbance was measured using a SPARK 10M spectrophotometer, and the wavelength was set to 550/600 nm. Results of protein secretion were presented as percent of the vehicle control.