Anti m csf

Neutralizing antibody anti-MCP-1, anti-M-CSF or both were added at day 0 and 3 at the concentration of 20 and 10 μg/ml, respectively (R&D Systems).

Cell supernatants were collected on day 3 and frozen until further use. Immunol 2HB microtiter plates (Thermo Scientific) were coated with anti-cytokine antibodies anti-IL-6 (Clone #6708), anti-IL-10 (Clone #127107), anti-TNFα (Clone #28401), and anti-M-CSF (Clone #21113) (R&D Systems, Minneapolis, MN, USA) and then blocked with PBS/2% BSA. Serially diluted standards and culture supernatants were added to these plates overnight. Plates were incubated with biotinylated anti-cytokine Ab (R&D Systems), followed by phosphatase-streptavidin (BD Biosciences) and K-Gold PNPP Substrate (Neogen Corporation, Lexington, KY, USA). Human IL-12p70 Quantikine, IL-4 Quantikine, and TGFβ1 Quantikine ELISAs were performed based on manufacturer’s instructions (R&D Systems). ELISAs were read using a SpectraMax M5 Microplate Reader and SoftMax Pro Acquisition and Analysis Software (both Molecular Devices, Sunnyvale, CA, USA).

After thawing, CD14+ monocytes were counted and resuspended in αMEM medium supplemented with 20% FBS. In a 96-well plate, we added 50 μL of cells in each well (45,000 cells/well) and then 50 μL of M-CSF, IL-34, and GM-CSF (all from R&D systems) diluted in 2% RPMI to obtain a 25, 50, and 10 ng/mL final concentration, respectively, or 50 μL of RA SF conditioned media. Medium (50 μL; 2% FBS) without any cytokine was used as control condition (CT). After 3 days, cell viability was assessed by WST-1 cell proliferation Reagent (Roche, France) by adding 10 μL per well. The formazan dye obtained from WST-1 cleavage by mitochondrial dehydrogenase was read by a microplate reader Wallac 1420 Victor 2 (Perkin Elmer, USA) between 420 and 480 nm after incubation for 4 hours at 37°C. Antibodies used to identify cytokines implicated in monocyte viability were as follows: anti M-CSF (R&D Systems), anti-IL-34 (Diaclone, INSERM UMR957), and anti-GM-CSF (R&D Systems) at 2, 10, and 5 μg/mL, respectively. We have previously tested different antibody concentrations to determine the most appropriate concentrations.