Pan t cell activation kit

Briefly, T cells were purified from peripheral blood samples by negative selection using the EasySep Human T cell Enrichment Kit (Catalog No. 17951, StemCell Technologies, Vancouver, Canada). Enriched T cells were stained with Cell Proliferation Dye eFluor 450 (Catalog No. 65-0842, ThermoFisher Scientific) to assess cell proliferation. Dye eFluor 450-labeled T cells (2.5 × 10⁴) were stimulated with anti-CD2/CD3/CD28 coated microbeads (Pan T Cell Activation Kit; Miltenyi Biotech, Bergisch Gladbach, Germany) or with uncoated microbeads as a negative control in a 1:10 bead:T cell ratio. These cells were cocultured with allogeneic CD106⁺ or CD106⁻ MSCs, which had been previously seeded in 96-well plates (5000 or 625 cells/well). The percentage of T cell proliferation was measured after 3.5 days in a Moflo XDP (Bechman, CA) and calculated by the percentage of FSC^highDye^low cells in the living cells gated by forward scatter (FSC) / side scatter (SSC). More than 8000 events were acquired for analysis. The data were normalized with respect to the percentage of activated T cells without coculture with MSCs.

Buffy coats were provided by the National Transfusion Centre of the Comunidad Autonoma of Madrid, Spain. Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation gradient using Ficoll-Paque Plus (GE Healthcare Biosciences AB, Uppsala, Sweden) following the supplier’s protocol.
PBMCs (2 × 10⁷) were resuspended in 10 mM of 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE, Molecular Probes, Eugene, OR, USA) solution and incubated at 37 °C for 10 min. Reaction was stopped by adding ice-cold medium (RPMI + 10% FBS) and cells were washed with ice-cold phosphate buffer saline. After resting overnight, CFSE-labeled PBMCs were cultured in 24-well plates alone or with Luci-eASCs (4 × 10⁴ cells/well; Luci-eASC:PBMC ratio 1:25) in a RPMI+10% FBS and were activated with the Pan T Cell Activation Kit (microbeads coated with anti-CD3, anti-CD2 and anti-CD28; Miltenyi Biotech, Bergisch Gladbach, Germany) following the manufacturer’s instructions [48 (link),49 (link)]. After 5 days, PBMCs were harvested, labeled with 7-AAD and anti-CD3 antibody and cell proliferation of the CD3⁺/7-AAD⁻ population (viable CD3 T lymphocytes) was determined by flow cytometry, according to the decrease in the CFSE fluorescence intensity. Data were analyzed with the use of FCSExpress 4 software (De Novo Software, Los Angeles, CA, USA) [18 (link)].