Chrompure

We removed SVF derived cells (SVFC) conditioned in GM/EGM from the culture at passage 3 by using 0.25% trypsin (Invitrogen). Cells were re-suspended in PBS, counted, then stained with Live/Dead Fixable Blue Dead Cell stain (Molecular Probes) according to manufacturer's recommendations. We then washed the cells twice with PBS and once with flow cytometry staining buffer (FCSB, 0.75% BSA, 1 mM EDTA, and 0.05% NaN₃ in PBS). Following these washes, the cells were incubated for 15 minutes at 4 °C with mouse IgG (ChromPure, Jackson ImmunoResearch) at 1 μg per 1 × 10⁵ cells to block non-specific and Fc receptor binding. After blocking, the cells were incubated for 25 minutes at 4 °C with the following antibodies at recommended concentrations: PE CD31 (clone LCI-4, Bio-Rad), APC CD105 (clone MEM-229, Novus Biologicals), FITC CD45 (clone K252.1E4, Bio-Rad), and PE-Cy7 CD90 (clone 5E10, BioLegend). The cells were then washed 2 times with FCSB and fixed with 3% paraformaldehyde for 25 minutes at RT in the dark. Following fixation, we washed the cells 2 times with FCSB before the final resuspension in FCSB, prior to analysis. The cells were analyzed on an LSR II (Becton Dickinson) within 18 hours of staining and fixation.

After 4 days of in vitro cultivation, cells were harvested and washed in PBS + 3% FCS. The antibodies (Abs) listed in Table 1 were used for cell surface and intracellular staining in FCM. The staining was performed in 96-well round bottom plates; for the cell surface staining three consecutive incubation steps were carried out: primary Abs, secondary Abs and a third incubation with mouse IgG molecules (2 μg per sample, ChromPure, Jackson ImmunoResearch, West Grove, PA, United States; blocking of the free binding sites of the secondary Abs) and the viability dye VDeFluor780 (Thermo Fisher Scientific). Each incubation lasted for 20 min at 4°C and was always completed by two washing steps with PBS + 3% FCS. Thereafter, the cells were fixed and permeabilized with eBioscience^TM Foxp3/Transcription factor staining buffer set (Thermo Fisher Scientific) and were then incubated for 30 min with the directly conjugated mAbs for the transcription factors (Table 1). After the last incubation, the cells were washed in Perm/Wash buffer included in the buffer set and analyzed in 200 μL of the same buffer by a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, United States). At least 2 × 10⁵ lymphocytes were recorded per sample. The obtained FCM data were further analyzed by FlowJo software version 10.5.3 (FlowJo LLC, Ashland, OR, United States).

To measure total IgG levels in the serum, plates were coated with 2 µg/ml of donkey anti-mouse IgG (Affinipure; Jackson ImmunoResearch Laboratories, West Grove, PA)–PBS, washed in PBS with 0.05% Tween 20, and blocked in 1% bovine serum albumin (BSA)–PBS prior to incubation with serial dilutions of serum or the mouse IgG standard (ChromPure; Jackson ImmunoResearch) in assay diluent (OptEIA assay diluent; BD Biosciences) overnight at 4°C. IgG was detected by the use of horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch), R & D Systems (Minneapolis, MN) substrate, and stop solution, and absorbance at 450 nm was read on a FilterMax5 microplate reader (Molecular Devices, Sunnyvale, CA). To measure levels of IgG specific for viral antigens, plates were first coated with 0.5% paraformaldehyde-fixed viral antigen–PBS overnight at 4°C.