Flow cytometry
For Th17 detection and sorting, the following antibodies were used:
APC/Cy7 anti-CD3
(HIT3a),
FITC
anti-CD4 (A161A1), PE anti-CCR4
(L291H4),
PE/Cy7 anti-CXCR3 (G025H7), PerCP/Cy5.5 anti-CCR6 (29-2L17), APC
anti-CCR10 (6588-5), PE anti-CD14 (63D3), PE anti-CXCR3 (G025H7), PE
anti-CCR10 (6588-5), PE/Cy7 anti-CCR4
(L291H4),
APC/Cy7 anti-CD27 (M-T271), Pacific blue anti-IL-17A (BL168), and
Pacific blue anti-GM-CSF (BVD2-21C11) were purchased from BioLegend. APC
anti-IL-17RE (FAB8358A) was purchased from R&D Systems. To stain cell
surface markers, PF mononuclear cells were incubated with 2 µg/ml of
each antibody for 30 min on ice before analysis and sorting on a BD
FACSAria Flow Cytometer (BD biosciences). To stain intracellular
cytokines after surface marker staining, cells were fixed with 200 μl of
2% paraformaldehyde for 20 minutes, followed by permeabilization in 1
ml of 90% methanol-PBS on ice for 25 min. Cells were then incubated
with 5 µg/ml of each antibody for 1 hour at room temperature,
respectively. Cells were washed and suspended in PBS for analysis on an
LSRII flow cytometer with a UV laser (BD biosciences).