Kaluza analysis 1.5 a software

Apoptotic cell death induced by the eluates of the tested compounds was assessed using a fluorescein isothiocyanate FITC Annexin V Apoptosis Detection Kit I (FITC Annexin V Apoptosis Detection Kit I, BD Bioscences, NJ, USA). Assays were prepared in 12 well plates by adding 500 μL (1 × 10⁶ cells/well) of complete medium and 500 μL of prepared eluates, and incubated for 24 h and 48 h. Cells treated with staurosporine (Sigma-Aldrich Corp., St. Louis, MO, USA) at a concentration of 1 μM for 16 h constituted a positive control. The negative control comprised cells suspended in the complete culture medium and incubated for 24 h and 48 h. Subsequently, cells were washed twice with cold phosphate buffered saline (PBS) (Sigma-Aldrich Corp., St. Louis, MO, USA) and then double stained with annexin V as a marker of early apoptosis, and propidium iodide (PI) as a marker of cell membrane disintegration, necrosis and late apoptosis. The percentage of apoptotic cells was calculated by flow cytometry (FC) using a CytoFLEX (Beckman Coulter, Brea, CA, USA). Obtained data were analyzed using Kaluza analysis 1.5 A software (Beckman Coulter) [33 (link)].

Apoptotic cell death induced by filtrates of test compounds was assessed using FITC Annexin V Apoptosis Detection Kit I purchased from BD Pharmingen™ (ApoAlert Annexin V, Clontech, California, USA). This method is based on high affinity of Annexin V to phosphatidylserine which, as a result of induction of apoptosis, is translocated to the outer parts of the cell membrane as well as on the propidium iodide (PI) that constitutes a marker of cell membrane permeability. SC cells (1 × 10⁶ cells/well) plated on 12-well plates were incubated with previously prepared compounds filtrates diluted in ratio 1:1 with medium for 24 h. Cells treated with staurosporine (Sigma-Aldrich Corp., St. Louis, MO, USA) at a concentration of 1 µM for 16 h constituted a positive control, whereas a negative control cells suspended in complete culture medium and incubated for 24 h. Subsequently cells were washed with cold PBS (Sigma-Aldrich Corp., St. Louis, MO, USA) twice and double stained with annexin V, as a marker of early apoptosis and PI as a marker of cell membrane disintegration, necrosis and late apoptosis. The percentage level of apoptotic cells was analyzed by FC using the Beckman Coulter CytoFLEX. The obtained data were analyzed using the Kaluza analysis 1.5 A software (Beckman Coulter).

The fluorescently conjugated antibodies were supplied by BD Biosciences (BD Biosciences, Erembodegen, Belgium), eBioscience (ebioscience, Schwerte, Germany), Biolegend (Biolegend, London, United Kingdom), Invitrogen (Invitrogen, Schwerte, Germany), and Beckman Coulter (Beckman Coulter, Krefeld, Germany). Following dyes were used for the Treg surface phenotyping: anti-CD4 (mouse IgG1, PerCP), anti-CD8 (mouse IgG1, APC-H7), anti-CD25 (mouse IgG1, PE-Cy7), anti-CD127 (mouse IgG1, FITC), and appropriate isotype controls. Cytokine assessment included: anti-CD3 (mouse IgG1, HorV450), anti-CD8 (mouse IgG1, APC-H7), anti-IL-2 (mouse IgG1, PE), anti-IL-10 (rat IgG1, APC), anti-IL-17A (mouse IgG1, PerCP), anti-IFNγ (mouse IgG1, FITC), anti-TNFα (mouse IgG1, PE), and anti-CD69 (mouse IgG1, PE-Cy7). We carried out flow cytometry acquisition on a 3-laser Navios instrument (Beckman Coulter), equipped to detect 10 fluorescent parameters. Compensation and data analyses were done with Kaluza Analysis 1.5a software (Beckman Coulter).

The analysis of the cell cycle was performed by FC using PI staining. Assays were prepared in 12-well plates by adding 1 × 10⁶ cells/well and after 24 h tested compounds were added at the appropriate concentrations and incubated for 48 h. The positive control constituted cells treated for 16 h with 1 μM nocodazole (Sigma-Aldrich Corp., St. Louis, MO, USA), whereas cells cultured in a complete medium for 48 h constituted a negative control. Afterwards, cells were washed twice with cold PBS (Sigma-Aldrich Corp., St. Louis, MO, USA) and then preserved with ice-cold 70% ethanol at − 20 °C for 20 min. Before staining with PI solution (10 μg/mL) (Sigma-Aldrich Corp., St. Louis, MO, USA) cells were processed with RNase A DNase & Protease-free (10 mg/mL) (Canvax Biotech, Córdoba, Spain) and incubated at 37 °C for 1 h. The percentage of cells in each phase of the cell cycle was assessed using Kaluza Analysis 1.5A software (Beckman Coulter, Brea, CA, USA). In the DNA content histograms, the x-axis showed the DNA content measured by PI fluorescence, while the y-axis indicated the number of cells^{30 (link),31 (link)}.

Mice were anesthetic and executed to harvest cerebrums. Cerebrums were dissected into small pieces and digested in papain for 30 min. After digestion, cell suspensions were filtered through 70 μm cell strainer to remove clumps, and cell pellets were resuspended in 30% Percoll solution following centrifugation. The upper myelin layer was discarded, and cell pellets were resuspended in medium (HBSS containing 2% FBS and 1 mM EDTA) to obtain single-cell suspension. Isolated single cells were treated as follows: 15 min incubation with FC-receptor blocker CD16/CD32 antibody (Thermo Scientific, MA5-18012, 1:200) at 4 °C, followed by 30 min incubation with anti-CD45-PE antibody (eBioscience, 12-0451-82, 1:200), anti-SIRPα-FITC antibody (Biolegend, 144006, 1:200), and anti-CD11b-APC antibody (eBioscience, 17-0112-82, 1:200) or rat IgG isotype. For intracellular staining, cells were fixed with 4%PFA for 10 min and permeabilized with PBS containing 0.1% Triton-X 100. Cells were incubated with anti-PSD95 antibody (Cell Signaling Technology, 3450, 1:500) for 30 min, then incubated with secondary fluorescent antibodies for 30 min. FACS analysis was performed on Beckman Coulter Gallios Flow Cytometer with Kaluza 1.0 software (Beckman Coulter). The data were analyzed by Kaluza Analysis 1.5a Software (Beckman Coulter).