Anti cd45 apc efluor 780 clone 30 f11

Six days post-infection, mice were exsanguinated and perfused as above; brains were harvested and homogenized in RPMI media-containing 0.5 mg/mL collagenase (Gibco LifeTechnologies), 0.01 mg/mL DNAse I (Roche) and 2 mM EDTA. Infiltrating cells were separated on a 33.3 % Percoll solution. Cells were stained for flow cytometry analyses with the following; Zombie Aqua Dye-V500 (BioLegend), anti-CD45-APC-eFluor780 (clone 30-F11, eBioscience), anti-CD4-PerCP Cyanine5.5 (clone RM4-5, eBioscience), anti-CD8alpha-PE (clone 53-6.7, eBioscience), anti-TCRbeta-FITC (clone H57-597, eBioscience), anti-CD19-BV421 (clone 6D5, BioLegend), F4/80-eFluor450 (clone BM8, eBioscience), anti-CD11b-APC (clone M1/70, eBioscience), anti-CD11c-PerCP Cyanine5.5 (clone N418, eBioscience), anti-Ly6C-PE (clone HK1.4, eBioscience), and anti-Ly6G-FITC (clone 1A8, BioLegend). Leukocytes were gated as CD45^hi cells.

BMMs from BALB/c WT mice were cultured in 1 × 10⁶ cells/well in 1 mL/well osteoclast medium with supplements on 24-well plates at 37 °C and 5.5% CO₂. BMMs were stimulated at day 0 of culture for 48 h with eosinophils (1:1 Eos/BMMs ratio), eosinophil supernatant (1:2 dilution), 10 µg/mL of MPO, and 0.05–0.1 µg/mL of EPX compared with unstimulated control. At day 2, the cells were detached from the plate by accutase and stained with MitoSOX^TM Red (Invitrogen; Cat# M36008) according to the manufacturer’s instructions. Afterwards, the cells were incubated with the following antibodies: anti-CD45 APC-eFluor™ 780 (clone 30-F11; 1:800; eBioscience; Cat# 47-0451-82), anti-CD11b FITC (clone M1/70; 1:400; BD Pharmingen; Cat# 557396), and anti-CD115 (CSF-1R) Brilliant Violet 421™ (clone AFS98; 1:800; BioLegend; Cat# 135513) in 1x PBS in the dark at 4 °C for 20 min. After washing, cells were resuspended in FACS buffer (1x PBS with 2% FBS and 5 mM EDTA) for flow cytometric analyses. Flow cytometry was performed on the Gallios^TM flow cytometer (Beckman Counter). Flow cytometry data were analyzed by Kaluza 2.1 (Beckman Counter).

Eosinophils were sorted from the blood and BM of IL-5tg/4get mice as described previously¹⁹ . The blood was lysed twice with 10 mL RCL buffer. BM cells were flushed out from the femur and tibia. Cells from blood and BM were pooled and put through 70 µm cell strainers. Cells were stained with anti-CD45 APC-eFluor™ 780 (clone 30-F11; 1:800; eBioscience; Cat# 47-0451-82), anti-CD125 (IL-5Rα) APC (clone REA343; 1:100; Miltenyi Biotec; Cat# 130-118-561), and anti-Siglec-F PE (clone E50-2440; 1:400; BD Pharmingen; Cat# 562068) in 1x PBS for 20 min in the dark at 4 °C. After washing, cells were resuspended in FACS buffer for sorting on MoFlo Astrios EQ (Beckman Counter). CD45⁺ CD125 ^int Siglec-F ⁺ granulocytes were sorted into 1x PBS with 2% FBS for in vitro experiments. Besides, 2 × 10⁶/mL sorted eosinophils were cultured for 48 h in αMEM and GlutaMAX (Gibco; Cat# 32571028) with 10% FBS (Gibco; Cat# A5256701) and 1% penicillin/streptomycin (Gibco; Cat# 15140122) at 37 °C and 5.5% CO₂ to generate eosinophil supernatant. After culture, the supernatant was separated from the cells by centrifugation and stored at −80 °C. The viability of the cultured eosinophils after 48 h was analyzed by flow cytometry with DAPI (Roche; Cat# 10236276001).