Cd3 percp

Cell surface markers of ATD-MSCs were analysed by flow cytometry on fresh SVF and on SVF derived after thawing of adipose tissue and its collagenase treatment. ATD-MSCs were identified as CD105, CD44, CD73, and CD271 positive cells and negative for CD45 expression. Standard labelling protocol was performed with the following antibodies fluorochrome-conjugated and isotypic controls: human CD105 PE (Invitrogen), CD73 FITC (kindly provided by Professor Malavasi, University of Turin), CD44 FITC, CD45 PerCP, CD3 PerCP, CD271 APC, IgG1 PE, IgG1 APC, and IgG2a PerCP (Miltenyi Biotec), and IgG1 FITC-conjugated (Immunostep). About 10⁵ events/sample were used for capture with CellQuest software. All data were analysed with FlowJo software (Tree Star).

To analyze the phenotype and purity of the fresh isolated products and expanded cells, they were stained with the ST-PE complex. Briefly, 0.75 μg of PE-labelled Strep-Tactin and 0.5 μg of antigen-specific MHC I-Strep (HLA-A*02:01/NLVPMVATV Streptamer) were incubated during 45 min at 4 °C in the dark to form the ST-PE complex. 0.2 μg of this reversible multimer were added to 1 × 10⁶ cells. The incubation was carried out during 45 min at 4 °C in the dark and afterwards cells were stained with, CD8-FITC (BioLegend, San Diego, USA), CD3-PerCP, CD137-APC (Miltenyi Biotec), and CD4-APC-Cy7 (BD Biosciences, San Jose, USA).
During the culture period, specificity and phenotype of expanded cells were analyzed every 7 days by staining with the ST-PE complex and monoclonal antibodies as previously described, with the addition of CD69 PE-Cy7 and CD57 VioBlue (Miltenyi Biotec).
Furthermore, at the beginning and the end of the expansion the memory phenotype of the cells was analyzed by staining with CD45RA-V450 and CCR7-PE-Cy7 (both BD Biosciences) during 15 min at room temperature.

Aliquots of 1 × 10⁶ isolated liver-derived cells and PBMC were stained for surface markers with pre-diluted fluorochrome-conjugated anti-human monoclonal antibodies: BD Multitest™ CD16-PE (IgG1, clone B73.1) and CD56-PE (IgG1, clone NCAM 16.2); CD3/CD16+CD56/CD45/CD19 reagent (CD3-FITC (IgG2a, clone SK7); CD45-PerCP (IgG1, clone 2D1); CD335/NKp46-PE-Cy7 (IgG1, clone 9E2/Nkp46); CD19-APC (IgG1, clone SJ25C1)) and CD3-FITC (IgG2a, clone SK7); CD4-PE-Cy7 (IgG1, clone L200); CD56-AlexaFluor-700 (IgG1, clone B159); CD8-APC-Cy7 (IgG1, clone SK1); antiCD337/NKp30-AlexaFluor-647 (IgG1, clone p30-15); and CD314/NKG2D-PerCP-Cy5.5 (IgG1, clone 1D11). These antibodies were purchased from BD Biosciences (Europe, dilution 1:10). CD161-FITC (IgG2a, clone 191B8); CD3-PerCP (IgG2a, clone BW264/56); CD16-APC (IgM, clone VEP13); and CD336/NKp44-PE (IgG1, clone 2.29) from Miltenyi Biotec (Bergisch Gladbach, Germany, dilution 1:20). TCRVa7.2-PE (IgG1, clone 3C10) was obtained from Biolegend (Campoverde s.r.l., Milan, Italy, dilution 1:25). At room temperature in the dark, LP samples were incubated for 30 min, and washed once with PBS/2% FBS. They were analyzed by flow cytometry with a FACSAria II cytometer (BD Biosciences, Eysins, Switzerland).

After five days in culture, surface staining was performed on monocyte-derived dendritic cells (MoDCs) for flow cytometry analysis with mouse anti-human HLA-DR FITC, CD83 PE, CD86 APC, CD54 PeVio770, CD14 FITC, CD3 PercP (all from Miltenyi Biotech) and analyzed by flow-cytometer LSRFortessa™ X-20 cell analyzer (BD Bioscience, Frankin Lake, NJ, USA) according to standard protocol. MoDC were then incubated with natural and synthetic compound in 12-well plates at concentration of 8 × 10⁵ cell/mL. Stimulation with PAM2CSK4 1 µg/mL (Invivogen, San Diego, CA, USA) was used as positive control. Cells treated only with vehicle (DMSO) were used as the control. For the experiment with the anti TLR-4 neutralizing antibody, cells were incubated 30 min with only the antibody (1 µg/mL) at 37 °C before addition of PGDG. After 24 h, expression of all surface markers was estimated again by staining with fluorochrome-conjugated antibodies.

PBMC were collected and aliquoted in 1 × 10⁶ cells/mL RPMI medium plus 10% Fetal Bovine Serum and then washed by centrifugation. The following anti-human monoclonal antibodies were added: CD3-PerCP, CD4⁺-APC-Vio770, CD8⁺-FITC, CD4⁺ 5RO-PEVio770, CD27-VioBlue, CD38-APC, (Miltenyi Biotec, Bergisch Gladbach, Germany). Samples were acquired by Miltenyi Biotec flow cytometer-MACSQuant Analyzer (8 fluorescence channels, 3 lasers). Gating and data analysis were performed using MACSQuantify software 2.5 (Miltenyi Biotec, Bergisch Gladbach, Germany).

Peripheral blood of patients affected by IL-17 correlated disease was drawn after their informed consent (see above). PBMCs were obtained after gradient stratification on Ficoll-Paque Plus (GE Healthcare); cells were stained with monoclonal antibodies anti CD3 Percp, CD4 Fitc, CD161 Pe (Miltenyi biotech) for FACS analysis; PBMCs mRNA was also extracted and reverse transcribed for qPCR Real Time analysis, as described below. Sera were obtained according to a standard protocol, by blood centrifugation, and S1P levels were measured as below described.