Luminex magpix system 40 072

Nonsilenced and ATP6V₀d2-KD macrophages were cultivated in 24-well plates in complete medium stimulated or not with IFN-γ/LPS or ox-LDL for 48 hours. Cell culture supernatants were collected and stored at -80°C until analysis. Nitric oxide concentrations from 25 μl of supernatants were assessed as described [99 (link)] using the chemoluminescence reader Nitric Oxide Analyzer (NOA 208i –Sievers). To determine cytokine concentrations, supernatants were loaded with the Milliplex Map Mouse Cytokine/Chemokine Magnetic Bead Panel and Milliplex Map TGFβ1 Single Plex Magnetic Bead Kit (MCYTOMAG-70K and TGFBMAG-64K-01, Merck Milipore), following the manufacturer’s instructions. Samples were then analyzed by Luminex MAGPIX System 40–072 (Merck Millipore). The NO and cytokine concentrations were normalized according to the macrophage protein lysate concentration, as assessed using the Bradford method.

After donation, blood samples were immediately spun at 2500 rpm for 15 minutes at room temperature, and 2-ml plasma aliquots with 40 μl of dipeptidyl peptidase-4 protease inhibitor (Merck Millipore, Billerica, MA, USA) were stored at −80 °C. All plasma samples were thawed only once for determination of the concentration of 13 soluble receptors (HSCR-32 K; Merck Millipore) and 64 cytokines (HCYTOMAG-60 K and HCYP2MAG-62 K; Merck Millipore) by addressable laser bead immunoenzyme assay according to the manufacturer’s specifications and read using the Luminex MAGPIX® System 40-072 (Merck Millipore). sCD40L plasma levels were subsequently assessed by enzyme-linked immunosorbent assay (ELISA) using the Human sCD40L Platinum ELISA kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s specifications and read at λ = 450/570 nm in a VICTOR X3 2030 multilabel ELISA reader (PerkinElmer, Singapore).