Aqua fluorescent live dead stain

Raw 264.7 macrophages were treated with IL-33 (1–100 ng) or deferoxamine mesylate (0.1–1.5 mM) for 24 h before being trypsinized and counted. Cells were then washed with 500 µL cell sorting buffer (CSB; 1× PBS, 2% newborn calf serum, 0.1% NaN₃, 5 mM EDTA, pH 8.0) and blocked for 15 min with an Fc block derived from a 2.4G2 hybridoma cell (ATCC, HB-197) supernatant. PerCP/Cy5.5 CD80 (BioLegend, cat# 104722, San Diego, CA, USA), PE-CD86 (BioLegend, cat# 159204), APC-CD40 (BioLegend, cat# 124612), PE-Cy7-IA/IE (BioLegend, cat# 107630), FITC IL-33R (MD Bioproducts, cat# 101001F, St. Paul, MN, USA), and aqua fluorescent live/dead stain (Invitrogen, cat# L34966) were added directly to cells following Fc block incubation for 30 min at 4 °C protected from light. Then, the cells were washed with CSB and fixed for 20 min at room temperature in 2% paraformaldehyde protected from light. The cells were washed again with CSB and resuspended in 500 µL fresh CSB for analysis via flow cytometry on a BD LSR Fortessa.

Anti‐CD4 (RM4‐5), anti‐CD45.1 (A20), anti‐CD45.2 (104), anti‐CD8α (53‐6.7), anti‐T‐cell receptor‐β (TCRβ) (H57‐597), anti‐CD44 (IM7), anti‐CD62L (MEL‐14), anti‐NK1.1 (PK136), anti‐granzyme B (GB11), anti‐IFN‐γ (XMG1.2), anti‐IL‐17A (TC11‐18H10.1), anti‐IL‐10 (JES5‐16E3), anti‐OX40 (OX‐86), anti‐GITR (DTA‐1), anti‐CD122 (5H4), anti‐ICOS (C398.4A), and anti‐LPAM‐1 (DATK32) antibodies for flow cytometry were purchased from BioLegend. Anti‐Foxp3 (FJK‐16s) was obtained from eBioscience. Fc block (2.4G2) and anti‐CTLA4 antibody (UC10‐4F10‐11) were obtained from Tonbo Bioscience, whereas anti‐IL‐2 antibody (JES6‐5H4) was obtained from Miltenyi Biotec. Aqua fluorescent LIVE/DEAD™ stain and carboxyfluorescein succinimidyl ester (CFSE) was obtained from Invitrogen. Cells were stained using LIVE/DEAD™ stain before surface antibody staining or intracellular staining for IFN‐γ, IL‐17, IL‐2, FoxP3, or CTLA4. LEN was obtained from FUJIFILM Wako Pure Chemical Corporation. A stock solution of LEN was prepared in dimethyl sulfoxide, stored at –80°C, and diluted with sterile phosphate‐buffered saline (PBS) immediately before the experiments were conducted.