Tetradecanoylphorbol Acetate

For flow cytometry analysis, 5 mice in each group were anesthetized. The lumbar spinal cord and lymph nodes were carefully dissected and dissociated at the time of the peak clinical score as previously described [23 (link), 33 (link), 34 (link)]. A single-cell suspension was prepared and fixed with 2 % paraformaldehyde for surface cell analysis. Cells were then washed with 2 % FBS in PBS, incubated with mouse anti-rat CD32 (BD Biosciences, San Jose, CA, USA) for 10 min to block the Fc receptor and washed twice with 2 % FBS in PBS. For surface cell analysis, cells were incubated with APC anti-mouse CD11b (OX-42; Biolegend, San Diego, CA, USA), PE anti-mouse CD45 (OX-1; Biolegend), APC anti-mouse CD4 (OX-35, BD Biosciences), and PE anti-mouse CD8a (OX-8, BD Biosciences) for 30 min at 4 °C. Microglia and macrophages were identified based on their relative CD45 expression levels [54 (link)]. Briefly, after acquiring unstained and single color control samples to calculate compensation matrix, we acquired 1 × 10⁴ events within the combined gate based on physical parameters [forward scatter (FSC) and side scatter (SSC)]. CD11b⁺/CD45^+(low) cells and CD11b⁺/CD45^+(high) cells were gated as resident microglia and macrophages, respectively [54 (link)]. For intracellular cell analysis, cells were restimulated with phorbol-12-myristate-13-acetate and ionomycin and Golgistop in RPMI media. After 5 h, cells were stained with PerCP-Cy 5.5 anti-mouse CD4 (RM4-5; BD Biosciences), FITC anti-mouse IFN-γ (XMG1.2; BD Biosciences), PE anti-mouse IL-17A (TC11-18H10; BD Biosciences), PE anti-mouse IL-4 (11B11; BD Biosciences), PE anti-mouse CD25 (PC61.5; FJK-16 s; eBioscience), and APC anti-mouse/rat Foxp3 (FJK-16 s; eBioscience) (Additional file 2) for 30 min at 4 °C. The cells were washed twice with 2 % FBS in PBS and used for flow cytometry. To identify CD4⁺ T cell populations, we first gated on cells (1 × 10⁴) based on FSC and SSC properties. CD4⁺ T cells were used to analyze populations of Th1, Th2, Th17, and Treg cells on CD4. Three-color staining was performed for one cell for simultaneous analysis. Intracellular cytokine results were indicated as percentages within the CD4⁺ population. Data were collected on a FACS Calibur flow cytometer (BD Biosciences) and analyzed using Cell Quest Pro software (BD Biosciences).

The human monocyte cell line THP-1 cells were purchased from ATCC. THP-1 cells were incubated in RPMI-1640 medium containing 10 mmol/L HEPES, 10% FBS and 1% penicillin/streptomycin antibiotic mixture, and then incubated for 24 h at 37 °C in a constant temperature incubator containing 5% CO₂. Subsequently, in order to induce THP-1 cells to become adherent macrophages, the cells were seeded into 6-well plates, and the culture medium involving PMA (phorbol 12-myristate 13-acetate) at a final concentration of 100 ng/mL was added, and the cells was incubated for another 24 h. For cell model construction, the cells were cultured in serum-free medium containing different concentrations of oxidized low-density lipoprotein (ox-LDL) for 24 h based on the previously published methods [14 (link)].
For cell transfection, according to the instructions of the kit, miR-218-5p mimic and mimic-negative control (mimic-NC) were transfected into THP-1 cells using Lipofectamine 2000 transfection reagent. The transfection efficiency was detected by RT-qPCR 6 h after transfection.