Ccr7 pe 150503

Thawed MNCs were stained with anti-CD45-APC-H7 (clone 2D1), -CD3-PeCy7 (SK7), -CD4-PerCP (SK3), -CD45RA-AlexaFluor700 (GB11) and -CCR7-PE (150503) (R&D Systems, Minneapolis, MN, USA) antibodies. The stained MNCs were acquired with FACSAriaII and analysed with FlowJo (Version 9.6.1, TreeStar). All antibodies were purchased from BD Biosciences unless mentioned otherwise.

The following fluorochrome-conjugated Abs were used for polychromatic flow cytometry analysis: CD3-Pacific Blue (UCHT1), CD4-Alexa700 (RPA-T4), CD45RA-APC-Cy7 (HI100), CCR6-PE (11A9), CCR7-PE-Cy7 (3D12), CD25-PE (M-A251), CD26-FITC (L272), CD127-AF647 (HIL-7R-M21), CD161-PE-Cy5 (DX12), IFNγ-Alexa 700 (B27), CCR5-PE (2D7), CXCR4-PE (12G5), and CD8-APC H7 (SK1) (BD Pharmingen), CD45RA-APC eFluor780 (HI100), CD56-FITC (MEM188), IL-17A-PE (eBio64DEC17), FoxP3-AF488 (PCH101), TNFα-Pacific Blue (Mab11), and IL-17A-eFluor660 (eBio64CAP17) (eBioscience), CD8-FITC (BW135/80), CD19-FITC (LT19) (Miltenyi), CCR7-PE (150503) (R&D) and CD31-BV605 (WM59) (Biolegend). Cell phenotype was analyzed by flow cytometry using the BD LSRII cytometer and BD Diva software. A viability staining Vivid (Invitrogen) was included in each staining cocktail to exclude dead cells from our analysis. FACS analysis was performed using the FlowJo software (^©Tree Star, Inc.). For multicolor analysis, all Abs were titrated for an optimal noise/signal ratio and Abs cocktails were validated by comparing single to multiple staining. Positivity gates were placed based on fluorescence minus one (FMO), as previously described [21 (link),101 ].

Murine antibodies with the following specificities and conjugations were purchased from Biolegend, CD11c-PeCy7, CD80-PE, CD86-APC, CD40-FITC, MHC-II PerCPCy5.5, together with appropriate isotype matched controls. After staining, cells were fixed with 1% paraformaldehyde in 0.85% saline and stored in the refrigerator prior to acquisition on the flow cytometer, within 48 hours.
Human monoclonal antibodies with the following specificities and conjugations were used: TLR2-FITC (TLR2.3), TLR4-FITC (HTA125), CD40-PE (LOB7/6), DC exclusion cocktail-PE-Cy5 (CD3 (S4.1), CD14 (TUK4), CD16 (3G8), CD19 (SJ25-C1), CD34 (581)) were purchased from AbD Serotec (Oxford, UK). CD83-PE (HB15e), CD80-FITC (L307.4), HLA-DR-FITC/APC (G46-6), CD86-FITC (24F), IL-10-APC (JES3-19F1), IL-12-PE (C11.5), IL-6-FITC (MQ2-13A5), IL-17-PE (SCPL1362), IFNγ-APC (25723.11), FoxP3-PE (259D/C7), CD4-PE (RPA-T4), CD3-PE/PeCy5/APC (UCHT1), CD8-APC (SK1), CD45RO-PE (UCHL1), CD45RA-PeCy5 (H1100) were purchased from BD Biosciences (Oxford, UK). CCR7-PE (150503) and TGFβ-PE (IC388P) were purchased from R&D Systems (Abingdon, UK). Appropriate isotype-matched control antibodies were purchased from the same manufacturers. After staining, cells were fixed with 1% paraformaldehyde in 0.85% saline and stored in the refrigerator prior to acquisition on the flow cytometer, within 48 hours.