All antibodies were used FITC, PE, Cy3, PE-Cy5, PE-Cy7, APC, eFluor660 or biotin conjugated. The biotinylated mAb were revealed with Streptavidin-PE-Cy5. mAb were used at 0.1–1 µg/1×10
6 cells in 100 µl PBS/2% FCS/0.02% NaN
3 (FACS buffer) or HBSS/0.3% BSA incubated on ice for 20–30 min. Antibodies used were: anti-CD16/CD32 (Fcγ III/II Receptor) (2.4G2), anti-CD90.1 (Thy1.1) (HIS51), anti-CD8α(Ly-2) (53–6.7), anti-CD3ε (145-2C11), anti-Vα2 T cell receptor (B20.1), anti-α4β7 (DATK32), anti-IA
b (AF6-120.1) and
anti-CD11c (HL3) all from BD Biosciences (Heidelberg, Germany), anti-CCR9 (B cell hybridoma supernatant, kindly provided by Prof. R. Förster),
anti-rat IgG (secondary for CCR9) (from Jackson ImmunoResearch Europe Ltd.), anti-CCR10 (248918), E-selectin/human IgG-Fc-Chimera (both from R&D) and
polyclonal rabbit anti-human IgG (secondary for E-lig staining) (DakoCytomation, Hamburg, Germany).
All stainings were performed following a standard protocol except of E-lig staining. This staining was performed as described [16] (
link). Control staining for E-lig and CCR9 was done with secondary Ab only, while corresponding isotype control mAb or fluorescence minus one (FMOs) were used as a control for all other stainings.
Data were acquired and analysed on a
FACScan using
CellQuestPro software (BD Biosciences) or on a
FACS Canto II (Becton Dickinson) and analysed using FlowJo.
Edele F., Dudda J.C., Bachtanian E., Jakob T., Pircher H, & Martin S.F. (2014). Efficiency of Dendritic Cell Vaccination against B16 Melanoma Depends on the Immunization Route. PLoS ONE, 9(8), e105266.