Nih 3t3 cells

To derive bone marrow-derived macrophages (BMDMs), bone marrow cells freshly isolated from the femora of Prep^-/- and Prep^+/- littermates were seeded in 6-well plates, and cultivated in RPMI 1640 supplemented with 10% FBS (Gibco, 16000-044), a penicillin/streptomycin mix (Gibco, 15140122) and 50 ng/ml M-CSF (PeproTech, AF-315-02) at 37 °C with 5% CO₂. BMDMs were fully differentiated and ready for use on Day 7. For macrophage polarization experiments, BMDMs were left unstimulated, stimulated with 50 ng/ml LPS, or stimulated with 20 ng/ml murine Interleukin-4 (PeproTech, AF-214-14) and 20 ng/ml murine Interleukin-13 (PeproTech, AF-315-02) for 24 h. RAW 264.7 cells were obtained from the China Center for Type Culture Collection (Shanghai, China) and cultured and maintained in DMEM supplemented with 10% FBS at 37 °C with 5% CO2. NIH-3T3 cells were purchased from Procell Life Science Technology Co., Ltd. (Wuhan, China) and were cultured and maintained in DMEM supplemented with 10% CS (AusGeneX, NCS-S) at 37 °C with 5% CO2.

The cytotoxicity of NNPs, BNPs, Tris-BNPs, IDB-loaded NPs (IDB/NNPs, IDB/BNPs, and IDB/Tris-BNPs), and free IDB was evaluated in mouse embryonic fibroblast cells (NIH/3T3 cells), B16 melanoma F10 cells (B16F10 cells), and human keratinocyte epithelial cells (HaCaT cells) (all from Procell Life Science & Technology, Wuhan, China). HaCaT (ATCC FS-0241) and NIH/3T3 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Grand Island, NY, USA) that contained 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin. B16F10 cells were cultured in Roswell Park Memorial Institute (RPIM) 1640 medium (Solarbio, Beijing, China) supplemented with 10% FBS and 1% penicillin/streptomycin.
NIH/3T3 and HaCaT cells were cultured in a 96-well plate at a density of 10,000 cells/well, with each well containing 100 μL of culture medium. B16F10 cells were plated at a density of 5000 cells/well. The cells were then incubated overnight at 37 °C. The next day, the medium was removed. A volume of 100 μL of DMEM, NNPs, BNPs, Tris-BNPs, IDB/NNPs, IDB/BNPs, IDB/Tris-BNPs, or free IDB in DMEM solution was added to the cells. The cells were incubated at 37 °C for 24 h. A CCK-8 assay kit (Dojindo, Kumamoto, Japan) was used to assess cell viability.

DSS (purity >98%; molecular weight 198.17), purchased from Meilunbio (Dalian, China), was dissolved in sterile distilled water as a 40-mM stock solution. NIH3T3 cells (Procell Life Science & Technology Co., Ltd., Wuhan, China) were cultured in Dulbecco’s modified eagle medium (DMEM, Gbico, USA) containing 10% fetal bovine serum (FBS) in a 37°C, 5% CO₂ incubator. Then, NIH3T3 cells were treated in different ways as followed: part of NIH3T3 cells were treated with DSS at different concentrations (0, 25, 50, 100, 200, and 400 μM) for 24 h; part of NIH3T3 cells were pretreated with various concentrations of DSS (0, 25, 50, 100, 200, and 400 μM) for 2 h, followed by stimulation with or without TGF-β1 (5 ng/mL) for 24 h and another part of NIH3T3 cells were pretreated with DSS (50 μM) for 2 h, followed by the stimulation with or without TGF-β1 (5 ng/mL) for 24 h.