Phorbol myristate acetate (pma)

THP1 cells were treated with 10 ng/mL phorbol 12-myristate 13-acetate (PMA, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) to induce the differentiation of the cells to macrophages [44 (link)]. The THP1-derived macrophages (1 × 10⁴ cells) were seeded onto an upper chamber of an 8 μm-pore transwell 24-well plate (BD Biosciences, NJ, USA), and supernatants or EVs (200 μg/mL) derived from different cells were added to the lower chamber. After incubation at 37 °C with 5% CO₂ for 24 h, the THP1-derived macrophages that migrated across the membranes were fixed with methanol, stained with 0.1% crystal violet solution, and counted under a Nikon 80i microscope (Minato-ku, Tokyo, Japan) [45 (link)].

Phorbol 12-myristate 13-acetate (PMA, Solarbio) and Ca-ionomycin (Solarbio) were added into the 12-well plates containing the SMC or TMC suspension, and brefeldin A (BFA, Solarbio) was added after 1 h followed by incubation for 3 h (total stimulation of 4 h). Cells were then collected, washed, and surface stained with APC-labeled CD4 antibody (Biolegend) at 4°C in the dark for 30 min. IC fixation buffer (eBioscience, USA) was then added followed by incubation at 4°C in the dark for 20 min. Finally, cells were washed, resuspended in permeabilization buffer (eBioscience), and stained with PE-labeled IL-17A antibody (Biolegend) at 4°C in the dark for 30 min. Flow cytometric analyses were performed using a FACScanto flow cytometer (BD, USA).

THP-1 cells and complete medium were purchased from National Collection of Authenticated Cell Cultures (China; Cat. No.: TCHu 57). THP-1 cells were inoculated in complete medium and cultured in incubator conditions of 37 °C, 5% CO₂, and 90% humidity. THP-1 cells (1 × 10⁶ cells /mL) were inoculated in 6-well plates and M0 macrophages were induced by stimulation with 100 ng/ml PMA (Solarbio, China) for 6 h. M0 macrophages were incubated with 20 ng/ml IFN-γ and 100 ng/ml LPS (Solarbio) for 48 h to induce M1 macrophages. M0 macrophages were incubated with 20 ng/ml IL-4 and 20 ng/ml IL-13 (Solarbio) for 48 h to induce M2 macrophages. Macrophages were collected, and CD68 and CD80, CD16 and CD86, CD163 and CD206 (BD Biosciences, San Jose, CA, USA) were detected using a BD-FACSAria™ Fusion flow cytometer (BD Biosciences, San Jose, CA, USA) to identify M0, M1 and M2 macrophages as described previously, respectively [19 (link), 20 (link)].

Human glioma cell lines U251MG, and TJ905 were purchased from the Chinese Academy of Sciences Cell Bank (China). Human glioma cell lines SNB19 and LN18 were purchased from the American Type Culture Collection (ATCC, US). The LNZ308 glioma cell line was generously provided by Prof. Huang of Tianjin Medical University General Hospital. Human glioma cell line TJ179 was isolated from human GBM tissue and cultured in nude mice following the protocol of Wang J et. al27 (link). Mouse glioma cell line GL261 was generously provided by Prof. Yao in Fudan University. The HMO6 microglial cell line was purchased from Beijing Future Biotechnology Co. (China), Ltd. All glioma and microglia cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, US) supplemented with 10% fetal bovine serum (FBS, Gibco, US). U937 monocytes were generously provided by Dr. Jin of Tianjin First Central Hospital and were cultured in Roswell Park Memorial Institute (RPMI) Medium 1640 (Gibco, US) supplemented with 10% fetal bovine serum (FBS, Gibco, USA). Phorbol 12-myristate 13-acetate (100 ng/ml, PMA, Solarbio, China) was added to stimulate U937 monocytes into macrophages. All cell culture media were supplemented with antibiotics (100 U/mL Penicillin-Streptomycin, Gibco, US) and incubated in 5% CO₂ at 37 ℃.

Freshly isolated neutrophils were treated with ox-LDL, PMA, ox-LDL plus PMA, respectively. As for PMA and ox-LDL plus PMA groups, neutrophils were pretreated with 1 nmol/L PMA (Solarbio, China) for 30 min and the cells were washed with RPMI1640 medium. Then the cells were incubated with 40 μg/ml ox-LDL (Yiyuanbiotech, China) for 12h, 24h, 36h, 48h, separately, and harvested for flow cytometric analysis.