Plasma cytokine quantification
The plasma levels of IL-5, IL-13, IL-6, IL-17A and TNF⍺ were quantified
using Quanterix Single Molecule-Arrays (SiMoA) analyser using the
Simoa™️ IL-5 Advantage Kit, Simoa™️ IL-13 Advantage Kit and Simoa™️
Cytokine 3-Plex B Advantage Kit respectively following the
manufacturer’s protocol.
Detection of cytokine
secretion by flow cytometry
Frozen cells were thawed using RPMI 1640 Medium (Gibco), supplemented
with 10% FBS (BioWhittaker) and washed with homemade 1X MACS buffer (1X
PBS + 0.5% BSA + 2mM EDTA). Cells were then pre-stained for 15
minutes at 37oC with 5%
CO2 using the following antibodies: anti-CD294 (clone
BM16, BD Pharmingen) and anti-CD45RB (clone MEM-55, ImmunoTools). Cells
were washed with 1X MACS buffer and then stimulated for 4 hours
at 37oC with 5%
CO2 with 20ng/ml Phorbol myristate acetate (PMA)
(Sigma-Aldrich), 1µM Ionomycin calcium
salt (Sigma-Aldrich) and GolgiPlug (BD Biosciences).
After 4 hours of stimulation, cells were washed with 1X PBS (Gibco)
and stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen)
for 10 minutes at room temperature. Cells were then washed with 1X MACS
buffer and stained for 15 minutes at 4oC using the
following antibodies: anti-CD161 (clone HP-3G10, eBioscience).
Intracellular staining was performed using the BD Cytofix/Cytoperm Kit
(BD Biosciences) according to the manufacturer’s instructions.
Cells were fixed with Fixation/Permeabilization solution (BD
Biosciences) for 20 minutes at 4oC. Cells were washed
and kept in 1X MACS buffer overnight at 4oC. On the
next day, cells were permeabilized with 1X Perm/Wash buffer for 15
minutes at 4oC and stained with the following
antibodies: anti-CD3 (clone UCHT1, BD Pharmingen), anti-CD4 (clone
RPA-T4, BD Pharmingen) and anti-IL-5 (clone TRFK5, BioLegend). After 30
minutes, cells were washed with 1X Perm/Wash buffer and resuspended in
1X MACS buffer. Samples were acquired using BD LSR II flow cytometer and
analysed using FlowJo v10 (BD).