7 aad viability dye

An aliquot of 1 × 10⁷ Jurkat cells in a round-bottom 2.0-mL microtube was fixed and permeabilized by exposure to two concentrations of Farmer’s solution [ethanol/acetic acid = 3:1 (v/v)] in PBS at 4 °C; 30% for 30 min and then 70% for 10 min. Cells were centrifuged at 600 × g for 5 min and washed with 2× saline sodium citrate (SSC). After centrifugation again, the cell pellet was resuspended in the mixture of 14 μL of CEP hybridization buffer, 2 μL of Vysis CEP 8 (D8Z2) SpectrumGreen Probe (Abbott Diagnostics, Lake Forest, IL), and 4 μL of Milli-Q, transferred to a 0.2-mL PCR tube, and then heated for hybridization at 80 °C for 5 min and 42 °C for 2 h. Cell suspension was diluted with 100 μL of 2 × SSC and centrifuged to form a pellet. The pellet was resuspended in 0.4 × SSC containing 0.43% of NP40 detergent and incubated at 72 °C for 2 min. The cells were harvested by centrifugation and then resuspended in 100 μL of 1% PFA in PBS. Before they were loaded into the VIFFI flow cytometer, the cells were stained by 1000× diluted 7-aminoactinomycin (7-AAD) Viability Dye [0.005% (w/v) as the original solution, Beckman Coulter, A07704] for counterstaining.

MLR potency assay was performed according to Nicotra et al. [16 (link)]. MLR assay was performed on UC-MSCs both in a resting state (NT) and after priming for 48 h with IFNγ, TNFα and IFNγ + TNFα. Briefly, peripheral blood mononuclear cells (PBMCs) pooled from 10 healthy donors and labeled with the CellTrace^TM Violet (CTV) Cell Proliferation Kit (Invitrogen, Ref C34557) were co-cultured with UC-MSCs at 0:1 (control), 1:10, 1:30, 1:100, 1:300 and 1:1000 UC-MSCs:PBMC ratio with a constant amount of PBMC (3 × 10⁵) and a decreasing amount of UC-MSCs from 3 × 10⁴ down to 3 × 10².
Cells were co-cultured in a culture medium composed of Roswell Park Memorial Institute (RPMI) Medium 1640, GlutaMAX^TM Supplement, HEPES (Gibco, Ref 72400-013), 10% human A/B serum (Eurobio, Ref CAEHUM010U), 1% Amphotericin B/Penicillin/Streptomycin (Gibco, Ref 15240062), and 10 UI/mL Heparin (PanPharma, Ref 5520508) at 37 °C, 5% CO₂ for 7 days. At day 4 ± 1, 50 µL of culture medium was added. At day 7, cells were labeled with 5 µL of human antibodies anti-CD3 PE (BD Biosciences, Ref 345765), anti-CD45 FITC (BD Biosciences, Ref 345808) and 7-AAD viability dye (Beckman Coulter, Ref A07704) for 15 min at 4 °C, protected from light. Cells were washed in PBS 1X before acquisition on the Attune NxT™ Thermofisher® Flow Cytometer and analyzed using Attune NxT™ software.

Enumeration of CD34+KDR+ EPCs was performed with three-color flow cytometry after direct immunolabeling of peripheral blood mononuclear cells (PBMCs) As previously described [10 (link)], PBMCs were isolated from 5 mL of heparinized peripheral blood by density gradient centrifugation with lymphocyte separation medium (PAA Laboratories, Pasching, Austria), and labeled with 10 μL of 7-AAD Viability Dye, 10 μL of FITC–CD34 antibody (Beckman Coulter), and 10 μL of PE–KDR antibody (R&D Systems, Abingdon, UK). 10μl of concentration-matched PE-conjugated murine IgG1 antibody was used as fluorescence minus one (FMO) control. After incubation for 30 min at room temperature, cells were washed, resuspended in 500 μL of phosphate-buffered saline (PBS) (Life Technologies), and analyzed with a FC 500 Cytometer. After selection of 7-AAD-negative cells, KDR+ cells were identified within CD34+ cells displaying FSC/SSC characteristics corresponding to the lymphocyte cluster. At least 5 × 10⁵ viable cells were acquired per run. The percentage of KDR+ cells among CD34+ cells was determined, and absolute values were calculated by multiplying the percentage of KDR+ cells by the absolute values of CD34+ cells. Absolute values of circulating CD34+ cells were determined in whole blood using the single plateform sequential gating strategy according to the standardized ISHAGE protocol [20 (link)].

Single-cell suspensions were obtained from mesenteric lymph nodes. The organs were minced and digested in Type IV Collagenase (3 mg/ml) and DNase I (50 mg/ml) in PBS + 5% FCS in a 37°C shaking water bath. After 45 min, digestion was stopped by adding cold PBS + 5 mM EDTA and single-cell suspensions were filtered through a 70-μm cell strainer and washed twice. For intracellular cytokine staining, cells were stimulated with PMA (50 ng/ml), ionomycin (2.5 μg/ml), and Brefeldin A (1 μg/ml) for 3 h in a 37°C, 5% CO₂ incubator. Cells were then stained with 7-AAD Viability Dye (Beckman Coulter) and incubated with 30% BSA and rat anti-mouse CD16/32 (eBioscience) for 10 min at 4°C to block Fc receptors. Subsequently cells were surface-stained with APC-Cy7–conjugated CD45 (clone 30-F11; BioLegend), BV785-conjugated CD3 (clone 17A2; BioLegend), BV510-conjugated CD4 (clone RM4-5; BioLegend), and Alexa Fluor 700–conjugated CD8a (clone 53-6.7; BioLegend) for 45 min at 4°C. Cells where then fixed, permeabilized using saponin (Sigma-Aldrich), stained for IFN-γ (PE, clone XMG1.2; eBioscience), acquired on BD LSRFortessa, and analyzed with FlowJo 10.8.2 software. Gating strategy was provided in Fig S4A.