Human il 13 quantikine elisa kit

IL-15 production was measured using a Human IL-15 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. IL-13 production was analyzed using Human IL-13 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA). For measuring IL-15 secretion upon T-cell activation, 5.0 × 10⁵ T cells were washed with PBS, resuspended in 275 µL of T-cell media (RPMI, 10% FBS, and 1% GlutaMAX) without cytokines and plated in 96-well, V-shaped plates. Cells were then activated with Immunocult™ Human CD3/CD28/CD2 T Cell Activator (StemCell Technologies, Vancouver, Canada) following the manufacturer’s protocol and incubated at 37 °C. After 24 h, 250 µL of media was collected for IL-15 ELISA from the wells and stored at −80 °C.

For cytokine secretion imaging using the LCI-S, we used anti-human IL-13 (MAB213 and BAF 213) and IL-4 (MAB604 and BAF204), anti-mouse IL-13 (MAB413 and BAF413) and IL-5 (MAB405 and BAM705) antibodies (R&D Systems, Minneapolis, MN, USA). For the measurement of IL-13 in culture supernatant, we used enzyme-linked immuno-sorbent assay (ELISA) kit (Human IL-13 Quantikine ELISA Kit, R&D Systems). For cell sorting of hILC2s, we used lineage antibody cocktails of human CD3 (UCHT1), CD14 (HCD14), CD16 (3G8), CD19 (HIB19), CD20 (2H7), CD56 (HCD56), PE-conjugated anti-human CD127 (A019D5), PE/Cy7-conjugated anti-human CD161 (HP-3G10) antibodies (BioLegend, San Diego, CA, USA), and Alexa Fluor 647-conjugated anti-human CRTH2 (BM16) antibody (BD Biosciences, San Jose, CA, USA).
For hILC2 stimulation, we used recombinant human IL-2 (Imunace35, Shionogi & Co., Ltd, Japan), IL-33, and TSLP (3625-IL/CF and 1398-TS/CF, R&D Systems, Minneapolis, MN, USA). For mILC2 stimulation, we used recombinant mouse IL-2 and IL-33 (402-ML and 3626-IL, R&D Systems).
For the miR-155 functional assay for hILC2 activation, we used miR-155 inhibitor (MISSION synthetic miRNA inhibitor, Sigma-Aldrich) and control (MISSION synthetic miRNA inhibitor negative control #1).