Intracellular Cytokine Staining for Th2 Cells

Intracellular staining to characterize primary Th2 cells was previously described in [42 , 43 (link)]. Briefly, cytokine staining for IL-4, IL-13 and IFNγ was performed following 4 h of stimulation with PMA (20 ng/mL) and ionomycin (1 μM) in the presence of Brefeldin A (10 μg/mL, all from Sigma Aldrich, Canada). Cells were fixed on ice (10 min) with paraformaldehyde (4%; Sigma Aldrich, Canada) and permeabilized on ice (10 min) with saponin (0.4%, Sigma Aldrich, Canada). Cells were incubated with at 4 °C (30 min) with IL-13-PE (Clone JES10-5A2, isotype rat IgG1κ PE), IFNγ-Alexa 647 (Clone B27, isotype mouse IgG1κ Alexa 647) or IL-4-Alexa 488 (Clone 8D4–8, isotype mouse IgG1κ Alexa 488) antibodies (BD Pharmingen, ON, Canada) and then washed with buffer. Fluorescence was assessed immediately using a FACS Calibur (Becton Dickson, ON, Canada) and data analyzed using FlowJo (Tree Star Inc., Ashland, OR, USA).