Human il 6 elisa kit

An in vitro ELISA for the quantitative measurement of human IL-6 in serum was performed using the human IL-6 ELISA kit (BD Biosciences, San Jose, CA, USA). The detection limit was 4.7pg/mL.

A human IL-6 ELISA kit (BD Biosciences, San Jose, CA, USA) was used to measure MonoMac-6 IL-6 production, as reported previously [47 (link)]. A multiplex human cytokine ELISA kit from Anogen (Mississauga, ON, Canada) was also used to evaluate interleukin (IL)-1α, IL-1β, IL-6, the tumor necrosis factor (TNF), monocyte chemoattractant protein-1 (MCP-1), interferon-γ (IFN-γ), and the granulocyte–macrophage colony-stimulating factor (GM-CSF) in MonoMac-6 cell supernatants, as reported previously [47 (link)].

Human IL-6 ELISA kit (Cat. 55522) and TMB substrate set (Cat. 555214) were purchased from BD Biosciences and used according to manufacturer’s instructions. Conditioned medium samples from senescent HF043 cells in a 384 well plate at ~ 1000 cells per well were diluted fivefold in DMEM-FBS prior to assay.

Total RNAs were extracted with Trizol (Invitrogen, NY, USA). After measuring the RNA concentration by using the NanoDrop ND-1000 spectrophotometer, 1 μg of total RNA was reverse-transcribed using cDNA synthesis kit (TaKaRa, Kusatsu, Shiga, Japan). GAPDH was used for an internal control. Primers used are as follows: 5′-AATCCCATCACCATCTTCCA-3′ (GAPDH F), 5′-TGGACTCCACGACGTACTCA-3′ (GAPDH R), 5′-AACCTTCCAAAGATGGCTGAA-3′ (IL-6 F), and 5′-CAGGAACTGGATCAGGACTTT-3′ (IL-6 R). Quantitative real-time PCRs were performed using SYBR green Master Mix (Takara, Shiga, Japan) in LightCycler 480 (Roche, Switzerland). Chromatin immunoprecipitation (ChIP) assays were performed using EpiSeeker ChIP kit (Abcam, Cambridge, UK) according to the manufacturer's instructions. In brief, cells were treated with SH003 for 3 hours and then fixed with 0.75% formaldehyde. Lysates were then sonicated and immunoprecipitated with anti-STAT3 antibody (Cell Signaling, Danvers, MA, USA). After reverse cross-linking, immunoprecipitated and purified DNA fragments were subjected to real-time PCRs. STAT3 binding region (−143 bp~48 bp) was amplified using primers as follows: F: 5′-GTTGTGTCTTGCCATGCTAAAG-3′, R: 5′-AGAATGAGCCTCAGACATCTCC-3′. ELISAs were performed with human IL-6 ELISA kit (BD Biosciences,San Jose CA, USA) according to the manufacturer's instructions.

FLS, HUVEC, SW982, and THP-1 cells were plated in 96 well plates (10⁴ to 10⁵ cells/well) for 48 h before treatment. At 2 days post-confluency, the cells were serum-starved and washed twice (with the exception of THP-1 cell, which were not washed) with FBS-free medium prior to the addition of test compounds. The cells were incubated in the presence or absence of test compounds or DMSO (vehicle control) for 30 min at 37°C in a 5% CO₂ atmosphere, IL-1β (5 ng/ml) was added, and the cells were incubated for an additional 24 h. Culture supernatants were collected and stored at –80°C. The levels of MMP-1 and MMP-3 were determined in culture supernatants using commercially available ELISA kits according to the manufacturer’s instructions (R&D System, Minneapolis, MN, USA). IL-6 levels were also determined in the supernatants using a human IL-6 ELISA kit (BD Biosciences, San Jose, CA, USA).