Mononuclear cells isolated from spleens, lymph nodes and tumours were first stained with a dead cell marker (LIVE/DEAD Fixable Aqua stain; Invitrogen) according to the manufacturer's instructions. Cells were then washed and stained for cell surface markers and chemokine receptors. Stained cells were washed, fixed and acquired on a FACS Canto II flow cytometer (BD Biosciences). Data analysis was performed using summit 4.3 software (Dako, Glostrup, Denmark). The antibodies used were as follows: anti-CD4-Pacific Blue antibody (BioLegend, San Diego, CA), goat polyclonal antibody to CCR1 (Santa Cruz Biotechnology, Santa Cruz, CA) followed with phycoerythrin (PE)-conjugated rabbit polyclonal antibody to goat IgG (Abcam, Cambridge, UK), rabbit monoclonal antibody to CCR2 (Abcam) followed with PE-conjugated donkey polyclonal antibody to rabbit IgG (Abcam), anti-CCR4-PE-Cy7 (BioLegend),anti-CCR3-Alexa Fluor 647 (BioLegend), biotin-conjugated anti-CCR5 antibodies followed with streptavidin-PerCP-Cy5.5 (both from eBioscience, San Diego, CA), anti-CXCR3-APC (eBioscience, San Diego, CA), anti-CXCR4-APC (BD Biosciences), rabbit polyclonal antibody to anti-CX3CR1 (Abcam) followed with PE-conjugated donkey polyclonal antibody to rabbit IgG (Abcam). Foxp3 expression was detected by GFP fluorescence.
Ondondo B., Colbeck E., Jones E., Smart K., Lauder S.N., Hindley J., Godkin A., Moser B., Ager A, & Gallimore A. (2015). A distinct chemokine axis does not account for enrichment of Foxp3+ CD4+ T cells in carcinogen-induced fibrosarcomas. Immunology, 145(1), 94-104.
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