Rmmfg e8

RAW264.7 cells were seeded at a density of 4 × 10⁵ per well on coverslips in 6-well plates, twenty-four hours post-seeding, RAW264.7 cells were pre-treated with or without 500 ng/ml rmMFG-E8 ((R&D Systems) for two hours, and then exposed to 10% CSE for 8 hours. After changing culture medium, FluoSpheres® Carboxylate-Modified Microspheres (Invitrigen, USA) were added to RAW264.7 cells at ratios of 1000:1. After co-culture for 90 minutes, unengulfed microspheres were removed by rising with PBS for three times, fixed in 4% paraformaldehyde, counterstained with DAPI to visualize nucleus. The phagocytic index was defined as the percentage of RAW264.7 cells engulfing one or more microspheres.

TNF-α, IL-1β, and IL-6 ELISA Kits were purchased from Biolegend (San Diego, CA,
USA). rmMFG-E8 was purchased from R&D Systems (Minneapolis, MN, USA).
Antibodies used for western blot were as follows: anti-Bcl-2 (Proteintech,
Rosemont, IL, USA), anti-Bax (Proteintech, Rosemont, IL, USA), anti-ß-actin
(Proteintech, Rosemont, IL, USA), and anti-histone H3 (Proteintech, Rosemont,
IL, USA). A NF-κB signal pathway kit was used to determine NF-κB pathway
activation (Cell Signaling Technology, Beverly, MA, USA). Blood urea nitrogen
(BUN) and serum creatinine (Cre) detection kits were purchased from the
Institute of Jiancheng Bioengineering (Nanjing, China). All other reagents were
of analytical grade.

RAW264.7 cells, obtained from American Type Culture Collection (ATCC), were seeded in 6-well plates in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. Twenty-four hours post-seeding, RAW264.7 cells were stimulated with 2.5%, 5%, 10% CSE, and harvested at indicated time points. To evaluate biological effect of MFG-E8, RAW264.7 cells were pre-treated with rmMFG-E8 (500 ng/ml) (R&D systems) for two hours followed by 10% CSE challenge for the subsequent experiments.