Bms223hs

Venous blood samples were collected from an antecubital vein into Ethylenediaminetetraacetic acid (EDTA) and serum separator tubes (SST) immediately before and after 7, 14, and 30 days of supplementation. One milliliter of EDTA blood was used to quantify erythrocyte sedimentation rate (ESR) by the Westegren method (Sedi-Rate, Globe Scientific, Inc., Mahwah, NJ). The remaining EDTA blood was stored at 4°C until centrifugation. SST tubes were stored at room temperature for 30 min then centrifuged with EDTA tubes for 15 min (2,500 × g) and 4°C after which supernatants were collected and stored at −80°C for later analysis. Two milliliter serum were sent to an outside facility (Pathology Laboratories, Inc., Toledo, OH) for measurement of uric acid (UA) and C-reactive protein (CRP). Remaining serum samples were assayed in house in duplicate for TNF-a (BMS223HS, Invitrogen, ThermoFisher Scientific, Vienna, Austria), insulin (Catalog #90095, Crystal Chem, Elk Grove, IL), glucose (Item #120003100P, Eton Bioscience, San Diego, CA), and glycated albumin (Catalog # IT3979, G-Biosciences, St. Louis MO). HOMA-IR was calculated as (insulin x glucose)/405 (30 (link)). Reference range for CRP was 0.00–0.744 mg·dL⁻¹ and 3.5 and 7.2 mg·dL⁻¹ for males and females for UA.

Patient serum and cell supernatant were taken to detect the levels of IL-1β, TNF-α, MMP-13 and other related factors following the instructions of ELISA kits (BMS224-2, BMS249-4, BMS223HS, EHMMP-13, Invitrogen). The sample to be tested, negative control and standard (positive control) were added to a 96-well plate before addition of the primary antibody. Primary antibody working solution was added to each well and shaken for 60 min, following which =the enzyme standard working solution was added and shaken for 60 min and 100 μL of substrate was further added for 10-min incubation without exposure to light. The termination solution was added, followed by the measurement of the optical density value at 450 nm with ELISA reader.

The protein levels of TNF-α, IL-6 and IL-1β secreted via HRCECs was measured using specific enzyme-linked immunosorbent assay (ELISA) kits (BMS223HS, BMS213-2 and BMS224-2, Invitrogen, USA) according to the manufacturer's protocol.

Levels of IL-17, IL-6; LIGHT; TNF-α and 27-OHC were quantified with human-specific enzyme-linked immunosorbent assay kits according to the manufacturers’ instructions (Cat# BMS2017HS [for IL-17]; Cat# BMS213HS [for IL-6]; Cat# BMS2218 [for LIGHT]; Cat# BMS223HS [for TNF-α] from Invitrogen and LS-F40084 [for 27-OHC] from LSBio).

The plasma concentration of TNFα, interleukin (IL)-6, IL-10, leptin, adiponectin, angiotensinogen, and irisin was measured using an enzyme-linked immunosorbent assay kit (eBioscience BMS223HS, BMS213HS, BMS215HS, Thermo Fisher Scientific, Waltham, MA, USA; Biovendor RD191001100, Brno, Czech Republic; Assaypro EA3501-1, St Charles, MO, USA; IBL International 27412, Hamburg, Germany; Biovendor RAG018R, Brno, Czech Republic). To determine malondialdehyde (MDA) levels, plasma samples were measured using the thiobarbituric acid-reactive substance (TBARS) method [17 (link)]. Briefly, we mixed samples with 20% acetic acid (pH 3.5), 0.8% thiobarbituric acid, and 8.1% sodium dodecyl sulfate and incubated the mixture at 95 °C for 1 h. After the mixture had cooled down to room temperature, trichloroacetic acid was added and detected spectrophotometrically at 532 nm.

ELISA assays were used on hPDL and OCM.30 cells to quantify the production of three inflammatory mediators: a growth factor, vascular endothelial growth factor A (VEGF-A), and two inflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interleukin 11 (IL-11), involved in inflammatory reaction periodontitis [24 (link),25 (link)]. These assays were made at 3 days following direct contact of each two-cell types with GX, GX-100, GX-200, Emd^®100, and Emd^®200. All cell lysates were subjected to ELISA for inflammatory mediators by applying ELISA kits: human (for hPDL) and mouse (for OCM.30) VEGF-A (KHG011, ThermoFisher Scientific), TNF-α (BMS223HS, ThermoFisher Scientific, France), and IL-11 (human EHIL11 and mouse EMIL11, ThermoFisher Scientific, France). Each ELISA was performed according to the manufacturer’s instruction. The experiment was performed in triplicate and repeated three times (n = 9); the data were compared to a standard curve. Absorption measurements were performed at 450 nm (Nanoquant infinite M200 pro, Tecan group Ltd., Lyon, France).