Phorbol 12 myristate 13 acetate

The SK-N-SH (Non-MYCN amplification) and SK-N-BE(2) cell line (MYCN amplification) (obtained from the Shanghai Institute of Biological Cell Research, Chinese Academy of Sciences) was cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS; Gibco) [22 (link), 23 ]. Additionally, the THP-1 cell line (also obtained from the Shanghai Institute of Biological Cell Research, Chinese Academy of Sciences) was cultured in 1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco) and 100 U/ml penicillin–streptomycin. HUEVC Human vascular endothelial cells were isolated by collagenase digestion of the human umbilical cord veins obtained from Children's Hospital Affiliated with Chongqing Medical University with the informed consents signed by donors. Human vascular endothelial cells were cultured in Endothelial Cell Medium (Lonza). 0.99 μm BKM 120 for SK-N-SH, 1.2 μm BKM 120 or SK-N-BE(2) (one of a PI3K inhibitor) (MCE)was used for the PI3K-AKT experiment [24 (link)]. For THP-1 cells, 100 ng/ml of PMA (Phorbol 12-myristate 13-acetate) (Multi-Science) was added, and recombinant human CXCL14 (MCE) was used for the experiment in vitro [25 (link)].

For preparation of immune cells in the CNS, brains, and spinal cords from MOG_35–55-immunized mice were excised and digested at 37°C with collagenase type IV (0.5 mg/ml; Sigma-Aldrich) and DNase I (10 U/ml; Roche) in RPMI 1640 under agitation (200 rpm) conditions for 60 min. The digested tissues were filtered through a 100-µm filter, and the plunger end of the syringe was used to push the cells over the filter. Homogeneous cell suspensions were centrifuged over the Percoll density gradient (GE Healthcare) and separated by collecting the interface fractions between 37% and 70% Percoll. Mononuclear cells were isolated from the interface. The cells were suspended in PBS containing 2% (wt/vol) FBS. After intensive washing, cells were stained with fluorochrome-conjugated surface marker antibodies for FACS analysis. The following antibodies were used: CD45, CD4, CD8, and CD11b. For intracellular cytokine staining, cells were stimulated with phorbol 12-myristate 13-acetate (Multi Sciences), ionomycin (Multi Sciences), and brefeldin A (Invitrogen) for the 4 h of culture. Cells were fixed and permeabilized with the Intracelluar Fixation & Permeabilization Buffer Set (eBioscience) and then subjected to cytokine staining flow cytometry analyses. All flow cytometry was performed on an Attune NxT flow cytometer (Thermo Fisher Scientific), and data were analyzed by FlowJo 7.6.1 software.

For preparation of immune cells, brains and spinal cords from MOG35-55–immunized mice were excised and digested at 37°C with DNase I (10 U/ml; Roche) and collagenase type IV (0.5 mg/ml; Sigma-Aldrich) in RPMI 1640 under agitation (200 rpm) conditions for 60 min. Single-cell suspensions were obtained by grinding through a 70-µm cell strainer. Subsequently, homogeneous cell suspensions were centrifuged over the Percoll density gradient (GE Healthcare) and separated by collecting the interface fractions between 37 and 70% Percoll. Mononuclear cells were isolated from the interface. After intensive washing, single-cell suspensions were stained with FVD eFluor 506, anti-CD45, anti-CD4, anti-CD8, and anti-CD11b for FACS analysis. For intracellular cytokine staining, cells were stimulated with phorbol 12-myristate 13-acetate (Multi Sciences), ionomycin (Multi Sciences), and brefeldin A (Invitrogen) for 4 h of culture. Cells were fixed and permeabilized with the Intracellular Fixation & Permeabilization Buffer Set (eBioscience) and then subjected to cytokine staining flow cytometry analyses. All flow cytometry was performed on an Attune NxT flow cytometer (Thermo Fisher Scientific), and data were analyzed by FlowJo 10.0.7 software. For FACS sorting, single cell suspensions were stained with FVD eFluor 506, anti-CD45, and anti-CD11b and sorted on a BD FACS Aria.