Il5 pe

CD4⁺CD62L⁺ T Cell Isolation Kit II, mouse (Miltenyi Biotec, 130-093-227); Naive CD4⁺ T Cell Isolation Kit, mouse (Miltenyi Biotec, 130-104-453); FITC BrdU Flow Kit (BD Pharmingen, 51-2354AK); LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit (Molecular probes, L34955); CellTrace™ Violet Cell Proliferation Kit (Molecular probes, C34571); Mouse IL-13 ELISA Ready-SET-Go Kit (eBioscience, 88-7137-22); Mouse IL-4 ELISA Ready-SET-Go Kit (eBioscience, 88-7044-88); Mouse IL-5 ELISA (BD Biosciences, 555236); PE Mouse anti-XBP1S Clone Q3-695 (BD Pharmingen, 562642); XBP1 (M-186)X- (Santa cruz, Sc 7160x); IL5-PE (BD Pharmingen, 554395); IL4-APC, Clone 11B11 (eBioscience, 17-7041-82); IL13-AF488, Clone eBio3A (eBioscience, 53-7133-82); IFNγ-Per CP Cy5.5, Clone XMG1.2 (eBioscience, 45-7311-82); FACS Staining buffer (eBioscience, 00-4222-26); IC Fixation buffer (eBioscience, 00-8222-49); Fixation/Permeabilization Diluent (eBioscience, 00-5223-56); Fixation/Permeabilization concentrate (eBioscience, 00-5123-43); Permeabilization buffer (eBioscience, 00-8333-56); SV Total RNA Isolation System (Promega, Z3101); Transcriptor High Fidelity cDNA Synthesis kit (Roche, 05081955001); SYBR™ Select Master Mix (Applied Biosystems, 4472908); Western blot antibodies: IRE1α (14C10) Rabbit mAb (Cell Signaling, #3294), IRE1 alpha [p Ser724] Antibody (Novus biologicals,NB100-2323).

For the intracellular cytokine staining, mouse spleen cells were stimulated with 2 μg/mL S1, S2 peptide pools spanning SARS-CoV-2 spike S1 and S2 respectively (15mers, overlapping by 11aa, GenScript, Nanjing, China) or equimolar amount of DMSO (negative control) in the presence of anti-mouse CD28 antibody (BD Bioscience, CA, USA) for 1 h. GolgiStop protein transport inhibitor (BD Bioscience) was added into the culture and further incubated for 5 h. After stimulation, cells were washed and stained with Fixable Viability Stain 510 (BD Bioscience). Cells were then blocked with anti-mouse CD16/32 and labeled with cell surface antibody cocktail including anti-mouse CD3-FITC, anti-mouse CD4-APC and anti-mouse CD8-Percp-cy5.5 (BD Bioscience) for 30 min at 4°C. Following surface staining, cells were incubated with fixing/permeabilizing solution (BD Bioscience) for 20 min at 4°C and stained with intracellular antibodies including anti-mouse IFN-γ-Pe-Cy7, IL-2-BV605, TNF-α-BV650, IL-4-BV711, and IL-5-PE (BD Bioscience) for 30 min at 4°C. Cells were washed and resuspended in PBS buffer and analyzed on a CYTEK Aurora/NL.

Fluorescent tagged antibodies used were specific for: CD3 allophycocyanin (APC), CD3 APC-cyanine7 (Cy7), CD4 phycoerythrin (PE), IL5 PE (BD Biosciences, San Jose CA); CD8 PE-Cy7 (Beckman Coulter Miami FL); IFN-gamma APC, FoxP3 (clone PCH101) and its isotype control (eBiosciences, San Diego CA); CD25-PE (Miltenyi Biotec Inc, Auburn CA).