Macsquant10 cytometer

All flow cytometry measurements were performed on a MACSQuant 10 cytometer (Miltenyi Biotec, Auburn, CA). Data analysis was performed with FlowJo (10.8.1). Cell viability was assessed by staining cells with Annexin V (Biolegend, CAT# 640945) and 4’,6-Diamidino-2-Phenylindole, Dilactate (DAPI) (Biolegend, CAT# 422801), viable cells were defined as cells staining negative for both DAPI and Annexin V. To compare flow cytometry data with mass data (Fig. 5 and Supplementary Fig. 9), subsets of 2500 cells were randomly sampled from each flow cytometry data set 1000 times to define a 95% confidence interval for cell viability readouts. These measurements were then compared to the control condition using a 3% limit of decision (described above for mass response measurements) to determine p values. To faithfully compare the magnitude of response between flow cytometry and mass readouts, the y-axis limit for mass response measurements was determined by finding the mass response between image events classified as “Cells” versus “Permeable” within the control population of cells measured for each experiment as a proxy for 100% viability loss as determined by flow cytometry (Supplementary Fig. 4).

Cells from two animals (IL17-eGFP mice for meninges and brain and C57BL/6 for NALT analysis) were combined and resuspended in 50 μl of FACS buffer (PBS, 2% FBS, 0.05% NaN₃) and incubated with 5 ng/μl of anti-CD16/CD32 antibodies (BioLegend, clone 93) for 10 minutes at 4°C to block nonspecific binding. Then, cells were stained for 15 minutes at 4°C with the following antibodies: CD45 (clone 30F.11, 2.5 ng/μl) CD11b (clone M1/70, 0.6 ng/μl), TCRβ (clone H57-597, 4 ng/μl), CD4 (clone RM4-5, 0.5 ng/μl), CD8 (clone 53–6.7, 2.5 ng/μL), CD19 (clone 6D5, 0.6 ng/μl), TCRγδ (clone GL3, 4 ng/μl) from BioLegend. Cells were washed with FACS buffer, resuspended in 200 μl of FACS buffer and samples were acquired on a MACSQuant10 cytometer (Miltenyi Biotec). Acquired data were analyzed using FlowJo software (Version 10, Tree Star).

For cell cycle studies, unsynchronized T47D cells were seeded at 250,000 cells/well in RPMI (Biowest, Nuaillé, France) 10% FBS medium supplemented with L-glutamine (2 mM), HEPES (10 mM), sodium pyruvate (1 mM) and PEST. Cells were treated with VEH (0.05% DMSO), 4-OHTAM (5 µM) or test compounds (5 µM) for 24 to 72 h. Cells were then washed in ice-cold PBS and fixed in cold 70% ethanol at −20 °C overnight. Then, the cell pellet was resuspended in FACs buffer (2 mM EDTA, 2% FBS in PBS) and incubated with Ki67-APC antibody (Miltenyi Biotec, Auburn, CA, USA) and DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich) for 20 min at room temperature. Finally, cells were washed and resuspended with FACs Buffer for cell cycle analyses. For apoptosis studies, an Annexin V-FITC Apoptosis Kit (Miltenyi Biotec) was used. Briefly, treated cells were collected in 100 μL of ice-cold AnnexinV binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) containing 10 μL of Annexin-V FITC and incubated for 15 min at room temperature. After that, cells were washed with 1 mL binding buffer, sedimented at 300× g 10 min, and resuspended with 500 μL binding buffer containing 5 μL of propidium iodide (PI; 100 μg/mL) prior to acquisition in MACSQuant10 cytometer (Miltenyi Biotec) where 10,000 or 20,000 events were used for cell cycle and apoptosis analysis, respectively.

The following mouse anti-human fluorescent-labeled antibodies were used for surface cell staining: CD56-APC-Vio770 (clone REA196), CD57-VioBlue (clone TB03), CD107a-APC (clone REA792), HLA-DR-PE-Vio770 (clone REA805), IFNγ-PE (clone 45-15), KIR2DL1-APC (clone REA284), KIR2DL2/DL3-PE (clone DX27), NKG2C-VioBright (clone REA205), HLA-E-PE (clone REA1031) (Miltenyi Biotec, Bergisch Gladbach, Germany); CD56-BrilliantViolet 421 (clone 5.1 H11), CD57-PE (clone HNK-1), CD57-APC (clone HNK-1) (Sony Biotechnology, San Jose, CA, USA); NKG2C-PE (clone 134591) (R&D Systems, Minneapolis, MN, USA).
PBMC/NK cells were washed in the PBA staining buffer (PBS containing 0.5% BSA (bovine serum albumin) (PanEco, Moscow, Russia) and 0.01% sodium azide (PanEco, Moscow, Russia) and incubated with antibodies for 30 min on ice in a PBA then washed twice in the same buffer before measurement. Flow cytometry analysis was carried out on a MACSQuant 10 cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) equipped with 405 nm, 488 nm, and 635 nm lasers.