Standard elisa kit

Human monocyte THP-1 cells (5 × 10⁵ cells/well) were seeded in 24-well culture plates. To study the cytokine modulations, media with different concentrations of DAR were added to the wells at concentrations of 0.10, 0.33, 1.00, 3.30, and 10.00 mg/ml. After 1-h treatment, 500 ng/ml LPS was added to the cells, except in control wells. Consumed media or cell supernatants were collected after 24 h to measure different cytokine levels such as TNF-α, IL-1b, and IL-6 using standard ELISA kits (BD Biosciences). Three biological and technical replicates were performed for each experiment according to the manufacturer’s instructions, and plates were read at 450 nm using Envision microplate reader (Perkin Elmer).

Media from control and LPS and/or AZD-treated Rin-5F cells was used for the measurement of inflammatory markers, TNF-α, and IL-6 using standard ELISA kits from BD Pharmingen (BD Biosciences, San Jose, United States) as described in the manufacturer’s protocols.
Cell lysates from LPS and/or AZD-treated cells were added to phospho-NF-κB p65 antibody-coated wells and incubated with NF-κB p65 antibody to detect the phospho-NF-κB p65 protein using the PathScan^® Phospho-NF-κB p65 sandwich ELISA assay kit as per the manufacturer’s protocol. Absorbance was read at 450 nm.
The peroxidase activity of the Cox enzyme was used as a basis for the measurement of cyclooxygenase -2 (Cox-2) activity in the cell lysates from control and LPS and/or AZD-treated cells. The assay was carried out in the presence of isozyme-specific inhibitors to distinguish Cox-2 from Cox-1 activity. The appearance of TMPD (N, N, N′, N′-tetramethyl-p-phenylenediamine) was monitored at 590 nm and used as a measure of Cox-2 activity.

After anesthetizing the mice with xylazine and Zoletil 50^® on day 8, BAL fluid sampling and tracheostomy were performed as previously described [32 (link)]. ROS level was determined using 2′, 7′-dichlorofluorescin diacetate (DCF-DA, Sigma-Aldrich, Carlsbad, CA, USA), according to previously reported protocol [33 (link)]. Additionally, TNF-α, IL-6, and MCP-1 levels were determined using standard ELISA kits (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s protocols. To calculate the number of inflammatory cells in BAL fluid samples, the samples were centrifuged using a CytoSpin 3 cytocentrifuge (Thermo Fisher Scientific, Waltham, MA, USA). Thereafter, the centrifuged preparations were stained on a slide using Diff-Quik^® reagent (SYSMEX, Kobe, Japan) and the inflammatory cells were counted using a light microscope at 400× magnification. The number of inflammatory celsl was determined as the average of counted cells in five different fields [16 (link)].

Concentrations of TNF-α and IL-10 were assessed in the supernatant from in vitro cell culture experiments using standard ELISA kits (BD Biosciences, San Deigo, CA) according to the manufacturer protocol. Results are presented for each cytokine (picogram/ml) following different treatments.