Rtnfα

RAW264.7 cells were obtained from Stem Cell Bank, Chinese Academy of Sciences. Mouse BMDMs were isolated from femur and tibia of C57BL/6 mice which were purchased from Institute of Shandong University Animal Experimental Center and differentiated into M0 macrophage by 25 ng/ml recombinant macrophage colony-stimulating factor (M-CSF) (R&D Systems, Minneapolis, MN, USA) treatment for 4 days. THP-1 cells (Stem Cell Bank, Chinese Academy of Sciences) were differentiated into M0 by 100 ng/ml PMA (Sigma, USA) treatment for 48 h. All cells were cultured in DMEM (Hyclone, Logan, UT, USA) containing 10% foetal bovine serum (FBS) (BioInd, Kibbutz, Israel) at 37 °C with 5% CO₂. When reached 80% confluence, cells were scraped, dissociated and counted and then plated in 6-well plates at a concentration of 2 × 10⁵/ml (in RAW264.7 cells) or 2 × 10^6/ml(in BMDMs and THP-1 cells). When reaching 60% confluence, cells were stimulated by 100 ng/ml P.g-LPS (InvivoGen, San Diego, CA, USA) with or without different doses of rPGRN (Sino Biological, Beijing, China) and TNF-α antibody (5 μg/ml; Abcam, Cambridge, UK) or a variety of concentrations of rTNF-α (Peprotech, Rocky Hill, NJ, USA) for 24 or 48 h, depending on different experimental goals. The normal medium containing equal amount of PBS to rPGRN and LPS groups was used as negative control.

Cell viability was measured by using Cell Counting Kit-8 (CCK-8, FDbio Science, Hangzhou, China) according to the manufacturer' instructions. Briefly, eEPCs were seeded in 96-well plates at 1 × 10⁴ cells/well in the presence or absence of r-TNFα (PeproTech Inc., Rocky Hill, NJ, USA). At the indicated time points, 10 μl of CCK8 solution was added into each well, followed by incubation of the plates at 39 °C in 5% CO₂ for 2 h. The optical density (OD) was measured at 450 nm.