C6 accuri system software

Cells (3 × 10⁵ cells/dish) were plated on 6-cm dishes for 24 h. The cells were then treated with various concentrations of CS for different of time intervals and then collected by centrifugation. The pellets were fixed with 75% ethanol at −20 °C overnight. Cells were then centrifuged and resuspended in 500 μL of PI staining solution comprising 2 mg/mL RNase, 1 mg/mL PI and 5% Triton X-100 maintained at 37 °C for 30 min in the dark. The cells were then analyzed using Accuri 5 flow cytometry (BD Biosciences, San Jose, CA). The percentages of cells distribution of the cell cycle in different phases (sub-G1, G0/G1, S, and G2/M) were calculated using C6 Accuri system software (BD Biosciences, San Jose, CA).

Sinularin- or sorafenib- treated cells were harvested, washed with PBS, and fixed with 70% ethanol at −20 °C overnight. The fixed cells were stained by propidium iodide (PI; Sigma-Aldrich, St. Louis, MO USA) containing RNase A (Sigma-Aldrich, St. Louis, MO USA) for 30 min in the dark at room temperature. The cells were then analyzed by an Accuri^TM C5 cytometer (BD Biosciences, San Jose, CA USA). Data were further analyzed by C6 Accuri system software (BD Biosciences, San Jose, CA USA).

All antibodies used for flow cytometry were from eBioscience (San Diego, CA, USA). To determine Tim-3 surface expression, primary microglia or BV2 cells (1 × 10⁵) were suspended in 100 μl of flow cytometry buffer (PBS containing 2% FBS), blocked with anti-CD16/32 (clone 93) for 10 minutes on ice, stained with anti-CD11b-APC (clone M1/70), and anti-Tim-3-PE (clone RMT3-23) or an isotype control (IgG2a) for 20 minutes. To determine if exogenous galectin-9 could enhance microglial expression of MHC II and CD86 following poly(I:C) stimulation, cells (2 × 10⁵) were plated onto sterile 35-mm petri-dishes (Falcon; Becton Dickinson, Franklin Lakes, NJ, USA) and treated with recombinant galectin-9 (2 μg/ml) poly(I:C) (50 μg/ml), or both for 24 hours. The cells were detached with ice-cold Hanks’ Balanced Salt Solution (HBSS) containing 5 mM EDTA, resuspended in flow cytometry buffer, blocked as described above then stained with anti-MHC II-PE (I-A/I-E; clone M5/114.15.2) or anti-CD86-PE (clone GL1) for 20 minutes on ice. Cells were gated on CD11b and the surface expression of Tim-3, MHC II or CD86 was determined by calculating mean fluorescence intensity using C6 Accuri system software (BD Bioscience, San Jose, CA, USA).

The mitochondria-specific cationic dye JC-1 (Invitrogen, Carlsbad, CA), which undergoes potential-dependent accumulation in mitochondria, was used to measure membrane potential. When the membrane potential (ΔΨ) is below 120 mV, JC-1 is monomeric and emits green light (540 nm) following excitation with blue light (490 nm). At membrane potentials higher than 120 mV, JC-1 monomer aggregates and emits red light (590 nm) following excitation with green light (540 nm). For the assay, cells were seeded onto 96-well plates and treated with various concentrations of CS for 24 h, followed by staining with 10 μg/ml JC-1 for 30 min at 37 °C. The samples were analyzed immediately using Accuri 5 flow cytometry (BD Biosciences, San Jose, CA). The data were analyzed using C6 Accuri system software (BD Biosciences, San Jose, CA) and displayed in a dot plot of JC-1 red fluorescence (Y-axis) against JC-1 green fluorescence (X-axis).

Spleens were harvested on the 33rd day and a single-cell suspension of splenocytes was generated by passing 50-mesh stainless mesh. For OVA-specific cell proliferation and cytokine expression, the cells (2 × 10⁶ cells/mL) were treated with OVA (100 μg/mL) in complete RPMI-1640 medium (Thermo Fisher Scientific, Inc.) and incubated for 72 h. The cells were collected and centrifuged at 1000× g for 15 min at 4 °C. The supernatant was collected for the detection of specific cytokine expression. The levels of IL-10, IL-4, and IFN-γ were evaluated by ELISA kits (Thermo Fisher Scientific, Inc.). For intracellular cytokine detection, splenocytes were incubated with OVA (50 μg/mL) for 72 h. Brefeldin A (BFA) (BD Biosciences, San Jose, CA, USA) was added for 4 h before harvesting, and then the cells were washed twice with PBS and stained with a PerCP-Cy5.5-conjugated anti-mouse CD4 antibody (BD Biosciences). Cells were fixed with fixation buffer (Biolegend, San Diego, CA, USA) and permeabilized with wash buffer (Biolegend) before staining with PE-conjugated specific antibodies against murine IFN-γ, IL-4, and Foxp3 (BioLegend). Subsequently, all samples were analyzed by a BD Accuri™ 5 flow cytometer (BD Biosciences). The mean fluorescence intensity (MFI) from each sample was calculated using C6 Accuri system software (version 1.0.264.15, BD Biosciences).

BD Accuri C6 Plus flow cytometer (BD, San Jose, CA) was used to identify MDSCs in the spleen and blood. BD Accuri C6 system software was used to analyze the data. Mouse MDSCs were identified by staining with the following panel of antibodies: fluorescein isothiocyanate-conjugated anti-mouse CD45 (30-F11, eBioscience, San Diego, CA), PerCP-Cy5.5-conjugated anti-mouse CD11b (M1/70, eBioscience, San Diego, CA), and phycoerythrin-conjugated anti-mouse Gr-1 Ly6G/Ly-6C (RB6-8C5, eBioscience, San Diego, CA). M-MDSCs (M-MDSCs/CD45⁺/CD11b⁺/Gr-1^low/mid) and granulocytic MDSCs (PMN-MDSCs/CD45⁺/CD11b⁺/Gr-1^high) were gated following the sorting strategies (Supplementary Fig. 2) to show the two distinct populations of MDSCs.

CD11c⁺ DCs were positively enriched from splenocytes using mouse CD11c MicroBeads UltraPure and LS separation columns (Miltenyi Biotec., San Diego, CA, USA) according to the manufacturer’s instructions. The purity of CD11c-positive DCs was above 80% after staining with FITC-conjugated mouse anti-CD11c antibody. The MFI of cells was calculated using BD Accuri™ C6 system software.