Rmst2 2

Antibodies against murine CD4 (RM4-5, AF700, BV650), CD8 (53-6.7, APC/Fire 750, BV785), CCR2 (SA203G11, BV421), CD11c (N418, APCCy7, Pe/Cy5), CD206 (C068C2, APC), CD11b (M1/70, BV421), F4/80 (BM8, BV510, BV711, FITC), TCRβ (H57-597, BV605), and NK1.1 (PK136, BV650) were purchased from BioLegend. Antibodies against CD4 (GK1.5, BUV395) and ST2 (U29-93, BV480) were purchased from BD Biosciences. Antibodies against B220 (RA3-6B2, AF488), CD11c (N418, e450, PE-Cy5.5), CD25 (PC61.5, PE-Cy7), CD4 (GK1.5, APC-eFluor 780, Super Bright 645), CD45 (30-F11, Pacific Orange), Foxp3 (FJK-16s, APC, e450, FITC), GITR (DTA-1, Super Bright 600), IL-10 (JES5-16E3, PE), KLRG1 (2F1, APC, APC-eFluor 780, PE-eFluor 610), NK1.1 (PK136, PE), and ST2 (RMST2-2, PE, PerCP-eFluor710) were purchased from eBioscience, Thermo Fisher Scientific. Viability dyes (e506, near IR, UV) were purchased from Invitrogen, Thermo Fisher Scientific.
Extracellular flow staining was performed in 2% FBS-PBS in the presence of BioLegend’s TruStain FcX for 30 minutes at 4°C. Fixation, permeabilization, and intracellular staining were performed using eBioscience’s (Thermo Fisher Scientific) Foxp3/transcription factor staining buffer set per manufacturer’s directions. Flow data were collected using a Cytek Aurora or BD Biosciences LSR and analyzed using FlowJo.

For the ILC2 analysis, the isolated MNCs were stained with a cocktail of the following antibodies: CD3e (145-2C11, BioLegend), CD11b (M1/70, eBioscience), CD11c (N418, BioLegend), CD45R (RA3-6B2, eBioscience), CD45 (30-F11, BioLegend), ST2 (RMST2-2, eBioscience), KLRG1 (2F1, eBioscience), and CD226 (10E5, BioLegend). ILC2s were characterized as a subpopulation of Lin (CD3e, CD11b, CD11c, and CD45R)⁻ CD45⁺ cells that expressed KLRG1 and ST2. For intracellular cytokine staining, the MNCs were stimulated with PMA/Ionomycin/BFA for 5 h. After extracellular staining, the cells were fixed, permeabilized, and incubated with anti-IL-5 (TRFK5, eBioscience) and anti-IL-13 (eBioBA, eBioscience) antibodies. For Th2 cell analysis, CD4 (RM4-5, eBioscience), CCR6/CD196 (29-2 L17, BioLegend), and CCR4/CD194 (2G12, BioLegend) antibodies were used. The Th2 cells were defined as CD4⁺ CCR4⁺ CCR6⁻ cells. Absolute counting beads (Invitrogen, USA) were employed to calculate the absolute cell number. FMO (Fluorescence Minus One) and matched isotype control were used as controls. The FCM data were obtained using a spectral cell analyzer (SONY SA3800) and analyzed with the Novoexpress software (Agilent Technologies).