Diva software

For the analysis of Th1, Th2, Th17 subsets, approximately 10⁶ leukocytes isolated from liver were stimulated with 20 ng/ml PMA, 1 μg/ml ionomycin and 2 mmol/ml monensin (Sigma-Aldrich, St. Louis, MO, USA) for 4 h at 37°C in 5% CO₂. For surface staining, the cells were collected and then incubated with anti-CD4-FITC for 30min. For intracellular staining, the cells were washed and fixed, permeabilized with Perm/Fix solution (eBioscience, San Diego, CA, USA) for 30 min and stained with anti- IFN-γ- Alexa Flour 488, anti-IL-4- APC and anti- IL-17A-PE (BD Biosciences) for 30 min for detecting Th1, Th2, Th17 cells, respectively. All samples were evaluated on flow cytometer (FACSCanto II; BD Biosciences, Franklin Lakes, NJ, USA) using Diva software (BD Biosciences).
For detection of Treg cells, the liver leukocytes were first surface incubated with anti-CD4-PerCP-Cyanine5.5 and anti-CD25-FITC for 30 min. Subsequently, Foxp3 staining buffer set (eBioscience, San Diego, CA, USA) were used to treat the cells and anti-Foxp3-APC were added for 1 hour. All samples were evaluated on flow cytometer (FACSCanto II; BD Biosciences, Franklin Lakes, NJ, USA) using Diva software (BD Biosciences). Samples were acquired and analyzed on a FACSCanto II flow cytometer.

For detection of surface Fas or CD74, cells were washed with 2% FBS/PBS and blocked with 0.025 mg/mL of mouse IgG blocking reagent (Invitrogen) at 4°C for 15 min in the dark and washed once with 2% FBS/PBS. Cells were then incubated with 3 μL of PE-conjugated anti-Fas antibody UB2 or with FITC-conjugated anti-CD74 antibody M-B741 (both from BD Biosciences) in 50 μL of 2% FBS/PBS at 4°C for 20 min in the dark. After washing cells 2× with 2% FBS/PBS, flow cytometry was performed on an BD LSRFortessa flow cytometer with Diva software (BD Bioscience).
For evaluation of apoptosis and cell death, cells were collected and resuspended in 1 mL of cold 1% FBS/PBS and stained with Vybrant® Apoptosis Assay Kit #5 according to protocol provided by the manufacturer (Molecular Probes). Flow cytometry was performed on BD FACSCanto II flow cytometer with Diva software (BD Bioscience). Data were analyzed using FlowJo software (Tree Star Inc.).

Mice were deeply anesthetized with isoflurane 72 h after reperfusion. Ischemic ipsilateral brains were dissected after transcardial perfusion with 100 ml of PBS. Brain leukocytes were collected as described previously [22 (link)]. In brief, ipsilateral hemispheres were homogenized and filtered through a 70-μm cell strainer. After centrifugation, the cells were resuspended in 7 ml FACS buffer, completely mixed with 3 ml of 90 % Percoll (GE Healthcare), and then 1 ml of 70 % Percoll was loaded under cell suspension. The cell suspension was then centrifuged at 500 g for 30 min at 4 °C. Leukocytes at the interphase were collected for immunostaining and FACS analysis. These cells were stained with differentially fluorochrome-labeled mAbs against TCR, CD4, and CD8 (AbD Serotec) on ice for 30 min to identify T cells, CD4+ T cells, and CD8+ T cells. Data on stained samples were acquired on a BD LSR II flow cytometer using Diva software (v6.1.2; Becton Dickinson, San Jose, CA, USA) and analyzed using FlowJo software (v7.6.2; Tree Star, Ashland, OR, USA).

Cell surface antigen expression was investigated in the control cell population, represented by AD-MSC-GFP and HOXB7-overexpressing AD-MSC. The following panels of monoclonal antibodies were assessed: APC-anti-CD45, PE anti-CD14, PE anti-CD73, and PE anti-HLA-DR (BD Pharmigen, San Diego, CA, USA); APC-anti-CD146 (MACS, Miltenyi Biotec Bergisch Gladbach, Germany); APC-anti-CD90 and APC-anti-CD105 (eBioscience, San Diego, CA, USA); and isotype controls (BD Biosciences and BioLegend, San Diego, CA, USA). Samples were analyzed using the FACSAria III flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The data were analyzed using DIVA software (Becton Dickinson, Franklin Lakes, NJ, USA). Cell cycle distribution was evaluated by propidium iodide (PI) staining. Single suspensions of 1 × 10⁶ AD-MSC-GFP or AD-MSC-HOXB7 at passage 12 were fixed with ice-cold 70% ethanol and stored at − 20 °C overnight. Cells were washed in PBS and resuspended in 200 μl of Cell Cycle Solution containing PI, RNase A, and Triton X-100 (Invitrogen, Molecular Probe, USA). The samples were then incubated for 30 min at room temperature in the dark and analyzed by FACSAria III and DIVA software.