, After 16 h of incubation, Treg cells were recovered, counted, and resuspended in RPMI-1640 medium with 2 mM L-glutamine, 10% heat-inactivated FBS, penicillin, and streptomycin. Previously isolated CD4 + CD25 ? cells were stained 15 min at 37°C with 1 ?M CellTrace? CFSE Cell Proliferation dye (ThermoFisher #C34554) at 1×10 7 cells per ml in PBS. Suppression assay was performed in U-bottom 96-well plates (Falcon, #353077) during 4 days at 37°C, 5% CO 2 , 20% O 2 : CFSE-stained Teff cells (1×10 4 cells/well) were incubated with Treg cells (Treg: Teff ratio = 1:1) pre-incubated with CAF-S1 or with medium only (control condition) and with anti-CD3/CD28 beads (Gibco, #11131D, 10 3 beads/ well). Wells with CSFE-stained Teff cells alone and wells with CSFE-stained Teff cells, in presence of anti-CD3/CD28 beads but no Treg cells, were used as a negative and positive control of proliferation, respectively. After 4 days, cells were stained with anti-CD4 antibody (see Supplementary Table 1 for references and conditions of dilution) and DAPI and analyzed on LSRFORTESSA analyzer (BD Biosciences). FITC fluorescence (corresponding to CFSE dye) was measured on DAPI ? CD4 + cells. The percentage of suppression was defined as: ((Log2(y) of Teff alone -Log2(y) of Teff + Treg)/Log2(y), with DAPI. After a washing step, DAPI ? CD4 + CD25 high CD127 ? CD45RA low cells (regulatory T cells, Treg) were sorted on FACSARIA (BD Biosciences) and pre-incubated overnight at 37°C, 5% CO 2 , 1.5%
, Twenty-four hours prior to transfection, 20,000 293T cells were plated in 96-well without antibiotics. Transient transfection was performed by mixing 0.15 ?l of DharmaFECT Duo Reagent (Dharmacon #T-2001-02) with 100 ng of 3?-UTR reporter plasmid and a respective final concentration of 10 nM of miRIDIAN microRNA Mimics, UTR luciferase vectors and luciferase reporter assays. Full-length human CXCL12? 3?-UTR luciferase reporter plasmid was bought from Switchgear Genomics
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