Hla dr v500

For surface staining, cells were incubated with antibodies diluted in PBS containing 2% FBS for 20 min, on ice. Conjugated antibodies to CD11c (FITC), CD124 (PE), CD14 (PERCP), CD80 (PE-Cy7), CD83 (APC), CD40 (APC-Cy7), CD86 (pacific blue) and HLA-DR (V500) were from BD Biosciences. Conjugated antibodies to CD4 (APC, APC-Cy7 and Alexa-Fluor 488), CD8 (PE and PERCP), CD25 (APC-Cy7) and CD127 (PE-Cy7) were from Biolegend. Intracellular cytokine staining was performed with BD Cytofix/Cytoperm kit (BD Biosciences). Cells were stimulated with Leukocyte Activation Cocktail with GoldiPlug (BD Biosciences) for 5 hours at 37°C in 5% CO₂, and were then stained with PE anti-IL-4 and APC anti-IFNγ (Biolegend). Regulatory T cells (CD4⁺CD127⁻CD25⁺Foxp3⁺) were stained for intracellularFoxp3 using the APC anti-human Foxp3 antibody kit (eBioscience). Flow cytometry was performed using a FACSCanto II (Becton Dickinson, Franklin Lakes, NJ), and theresults were analyzed with FlowJo 8.7 software.

For flow analysis, 5 × 10⁵ cells were stained in 100 μl staining buffer (PBS, 1% BSA) for 30 minutes at 4°C. Events were acquired on an LSRII flow cytometer (BD Bioscience, San Jose, CA) and analysis was performed in FlowJo 9.5.2 (Treestar Inc., Ashland, OR). Antibodies used for flow cytometric analysis were: CD80-FITC (BD), CD83-BV421 (BioLegend, San Diego, CA), HLA-DR-V500 (BD), CD11c-PECy7 (BD), CD11c-BV605 (BioLegend), PD-L1-FITC (clone M1H1, BD), B7-DC (PD-L2)-PerCPEFluor710 (eBioscience, San Diego, CA) and IDO-PE (clone 700838) (R&D Systems, Minneapolis, MN).

In order to analyze DCs subsets, 2 mL blood samples were collected in EDTA tubes from control subjects and patients with T1D at diagnosis, and at first and second year of disease progression. Blood samples were lysed (Lysing Buffer, BD Biosciences, San Jose, CA, USA) and stained with antibodies to CD45-AF700, CD3-APCH7, CD19-APCH7, CD14-V450, CD16-APC, CD11c-PECy7, CD123-PerCPCy5.5, CD56-PE, HLA-DR-V500, and Slan-FITC (BD Biosciences). A minimum of 10,000 events per sample was acquired using FACS LSRFortessa (BD Biosciences). Percentage and absolute counts (cells/μL) were analyzed using PerfectCount Microspheres (Cytognos SL, Salamanca, Spain) and FACSDiva software (BD Biosciences) following gating strategies based on international consensus (24 (link)).

For phenotypic analysis of NK cells, PBMC were stained with the following fluorochrome conjugated antibodies or isotype matched controls: CD3-Cy5.5/PerCP or CD3/Pe-Cy7, (eBioscience, Hatfield, UK), CD56-PE Texas Red (Beckman Coulter, High Wycombe, UK), CD56-FITC, CD16-APC Cy7, HLA-DR V500, CXCR3-Cy.5./PerCP, CD57-BV605, NKp46-V450, TRAIL-PE, (BD Biosciences, Oxford, UK), TRAIL-BV421, CD62L-AF700, CXCR6-PE, CD69-APC, CCR7-BV421, KLRG1-FITC, (Biolegend, London, UK), NKG2A-APC, NKG2C-Cy5.5/PerCP, NKG2D-PE (R&D systems, Abingdon, UK), NKp30-APC, NKp44-PE (Miltenyi Biotec, Surrey, UK), in the presence of fixable live/dead stain (Invitrogen, Paisley, Scotland). For phenotypic analysis of T cells PBMC were stained with the fluorochrome conjugated antibodies or isotype matched controls: CD3/Pe-Cy7, CD8-AF700, CD4-APC Cy7 (eBioscience, Hatfield, UK), CD38-PE Texas Red, CD14-V500, CD19-V500 (Biolegend, London, UK). Flourescence minus-one (FMOs) were used for gating purposes for all flourochromes; an example of the gating strategy is shown in S1A Fig. Cells were acquired on a FACS LSRII multicolour flow cytometer (Beckton Dickinson) and analysed using Flow Jo analysis software (Tree star, Ashland, OR, USA). In addition to percentage, absolute numbers of NK cell subsets were calculated by multiplying their percent by total lymphocyte count.

Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using Ficoll-Hypaque (Amersham Biosciences, Amersham, Buckinghamshire, UK) from 20 mL venous blood samples collected in EDTA tubes. Freshly isolated PBMCs at 1 × 10⁶ per tube were stained as previously described [12 (link)]. The following monoclonal antibodies were used for T cell immunophenotyping: CD3-APC, CD4-APC-CY7, CD45RA-PE-CY7, CCR7-PERCP-CY5.5 and HLADR-V500 (BD Biosciences, San Jose, CA, USA). After antibody staining, the cells were incubated with 200 µL of 100 nM MitoLite™ Orange FM at 37 °C for 15 min, and washed twice with phosphate-buffered saline. After surface staining with antibodies and mitochondrial staining with MitoLite, Caspase 1-FITC was used for intracellular staining. The isotype control of FITC, PE-CY7, V500 and PERCP-CY5.5 was performed for each experiment to determine gates for them.

For immunoflourescent staining of tissue sections the following primary antibodies were employed: CD68, 1 µg/ml (clone: PGM-1, Dako Cytomation, Hamburg, Germany); CD14, 1 µg/ml (clone: TÜK4, Dako); CD86, 10 µg/ml (clone: FUN-1, BD Pharmingen, Heidelberg, Germany). Isotype- and concentration-matched control antibodies (Dako) served as negative controls.
For flow cytometric analysis the following mAb were purchased from BD Bioscience (Heidelberg, Germany): CD3 V450, CD14 FITC, CD33 PE-Cy7, CD86 AF700, HLA-DR V500, CD117 APC.

The phenotype of CD34⁺ HSC exposed to betamethasone or fluticasone after 20 h of culture was determined by flow cytometry. To that end, 20.000 CD34⁺ cells of each group (control, betamethasone, and fluticasone) were stained with anti-human CD34 PE (BD Biosciences), HLA-ABC FITC (Immunotools), CD45 PercP (BD Biosciences), CD40 APC (Immunotools), CD54 PE-Cy7, CXCR4 APC-Cy7 and HLA-DR V500 (BD Biosciences), and median fluorescence intensity values were obtained using FACS Canto II cytometer (BD Biosciences). Data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).