L23105

B16 cells-expressing ovalbumin (courtesy of Lea Eisenach, Weizmann institute of Science) were suspended in DMEM medium w/o phenol red, supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, 100 mg/ml of streptomycin, 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, and 50 mM β-mercaptoethanol (Biological Industries). Cells were seeded 50–100 × 10³ B16 cells per well in a 24-well plate; 150–300 × 10³ OT-I T-cells pre-activated and cultured for 7 days, were added on top of the B16 cells. After 24–48 h, cells were collected, and a single-cell suspension was prepared and stained with live/dead stain (L23105, Life Technologies, Carlsbad, CA, USA), CD8 (BLG100734, Biolegend), FasL (BLG106606, Biolegend), and PD-1 (BLG135225, Biolegend). Cells were then fixated (BLG420801, Biolegend), permeablized (BLG521002, Biolegend), and stained for intracellular granzyme B (BLG515406, BioLegend). The mean fluorescent intensity of Granzyme B, FasL, and PD-1 of live CD8⁺ cells was measured and analyzed using flow cytometry (Becton Dickinson; FlowJo software).

Single-cell suspensions were obtained from peritoneal exudate cells (PECs) by peritoneal wash with 5-mL ice-cold phosphate buffer solution (PBS). PECs pellet were washed twice with complete Roswell Park Memorial Institute (RPMI) 1640 media and filtered with 70 μm nylon mesh (Fisher Scientific). ACK (Ammonium-Chloride-Potassium) Lysing Buffer (2-mL; GIBCO, Life technologies) was added to lyse red blood cells. Harvested cells were incubated in saturated doses of anti-mouse Fc receptor in 100-μl ice-cold FACS buffer (1% bovine serum albumin/0.01% sodium azide (NaN₃) in PBS) for 15 min. After washing, 3 × 10⁶ cells were stained with various combinations of antibodies in ice-cold FACS buffer for 15 min, and further collected on a LSR II cytofluorometer (Becton Dickinson, BD). Cells were gated according to size (SSC-A) and forward scatter (FSC-A). Blue-fluorescent reactive dye L23105 (Life Technologies) was used to discard dead cells. Absolute cell numbers were calculated with the total cell count multiplied successively by the percentages for the appropriate gates obtained through analysis in FlowJo Software (FlowJo, LLC).

Flow cytometric analysis was performed as previously described [19 (link)]. Briefly, harvested BMDM were incubated in 1 μg/mL of anti-mouse Fc receptor antibody in 100 mL PBS containing 0.5% BSA plus 0.02% NaN₃ (FACS buffer) for 15 min on ice. Subsequently, single-cell suspensions were stained for 15 min at 4°C with blue-fluorescent reactive dye, L23105 (Life Technologies) to discriminate dead cells. After washing, 1-3 × 10⁶ cells were surface-stained in FACS buffer for 15 min at 4°C with antibodies recognizing CD11b (Alexa Fluor 700, BioLegend), F4/80 (Brilliant Violet 785, BioLegend), CD86 (Brilliant Violet 421, BioLegend), PD-L1 (PE-Cy7, BioLegend), and PD-L2 (PE, BioLegend). Surface-stained cells were washed three times with FACS buffer and treated with Fix/Perm reagent according to the protocol of the cytofix/cytoperm kit (BD Biosciences, San Jose, CA, USA). The cells were intracellularly stained in FACS buffer containing anti-Nos2 (PE, eBiosciences) and anti-h/m arginase 1 (APC, R&D systems) for 30 min at 4°C and further collected on an LSR II cytofluorometer (BD, Franklin Lakes, NJ). Stained cells were gated according to size (SSC-A) and forward scatter (FSC-A) to eliminate debris. Doublets were excluded from the analysis by using forward scatter height (FSC-H) and FSC-A. Data analysis was performed using FlowJo Software (FlowJo, LLC).

Cells were plated at 700000 cells/T25 flask and grown for 12 days before exposure to CM for 24 h. Cells were detached using PBS EDTA and accutase. For flow cytometry there were 25000 cells per sample and for Image stream analysis there were 50000 cells per sample. Antibodies were used for the detection of astrocytes (GFAP 1:500 Millipore or GLAST 1:50 eBioscience), neurons (MAP2 1:2000 Synaptic systems 188 004 or CD90 1:200 eBioscience), autophagy (LC3 1:200 MBL and LysoID 1:500 Enzo Life Science) and a live/dead marker (1:2000, L23105 Life Technologies) and cell death (Annexin V 1:50, Life Technologies L23105). Mitochondrial function was accessed using Mitotracker Green FM 150 nM M7514, NaO 100 nM A1372, Mitosox 5 μM M36008 (all available from Life Technologies), and MitoID 1:2500 ENZ-51018, (Enzo Life Sciences). Cells were labelled for cell markers and mitochondrial functional indicators and the level of NaO, mitochondrial membrane potential, mitochondrial oxidative stress, and mitochondrial number. Cells from each exposure were compared for statistical difference from control. Flow cytometry was carried out on the BD LSRII and analysed using FloJo v 8.8.6. Cells prepared for detection of autophagy were run through Imagestream and analysed using IDEAS 6.1.303 software.