Pe cf594 anti cd4

Large intestine was shaken in 5 mM EDTA/1 mM DTT for 10 min and then transferred to fresh 5 mM EDTA/1 mM DTT to incubate for 10 more minutes to obtain IEL fractions. IEL pellets were mixed in 30% isotonic Percoll and layered onto 70% isotonic Percoll. Cells were collected from Percoll gradients following centrifugation at 1800 rpm for 20 min and washed in RPMI-1640. Cells were stained with a combination of the following fluorescence-conjugated mAbs: PE–CF594 anti-CD4 (BD), PE-Cy7 anti-TCRβ (eBioscience), PE anti-CD326 (EpCAM) (Miltenyi Biotec), eFluor450 anti-CD8β (eBioscience), eFluor450 anti-TCRγδ (eBioscience), APC anti-IFNγ (eBioscience), FITC anti-CD45 (eBioscience), PerCP-Cy5.5 anti-CD8α (eBioscience), BV650 anti-IL-17 (BD), APC anti-IL-22 (BioLegend). Dead cells were excluded with Fixable Aqua Dead Cell Stain kit (Invitrogen). Samples were acquired on LSR Fortessa II and analyzed with FlowJo software (v10.0; TreeStar).