Anti cd45 ef450 clone 30 f11

9464D-GD2 tumors were extracted on day 13 and incubated for 30 min at 37 °C in dissociation solution containing HBSS supplemented with 5% FBS, 1 mg/mL collagenase type D, and 100 μg/mL DNase I (Sigma-Aldrich) as previously described [12 (link)]. For cell surface staining, cells were incubated with anti-GD2-APC (clone 14G2a; BioLegend), anti-CD45-eF450 (clone 30-F11; eBioscience), anti-CD3-Alexa700 (clone 17A2; BioLegend), anti-CD4-PE-Dazzle594 (clone GK1.5; BioLegend), anti-CD8a-APC-eFluor780 (clone 53–6.7; eBioscience), anti-CD11b-BB700 (clone M1/70; BD Horizon), anti-Ly6G-BV711 (clone 1A8; BioLegend), anti-CD25-BB515 (clone PC61; BD Horizon), anti-FoxP3-PE-Cy7 (clone FJK-16 s; eBioscience), and Ghost Dye Violet 510 (Tonbo Biosciences). Flow cytometry data were acquired using an Attune NxT Flow Cytometer and analyzed using FlowJo version 10.1.

Flow cytometry and FACS were performed as described previously [27 (link),29 ]. Briefly, cells isolated from lung tissue as described above were pelleted and resuspended at 10⁷ cells/mL in PBS with 0.1% BSA. Non-specific antibody binding was blocked with anti-CD16/CD32 followed by a master mix antibody cocktail (50 μL per 10⁷ cells) that included 10 μL of each of the following antibodies: anti-CD45-eF450 (clone 30-F11; eBioscience), anti-CD11c-AF700 (clone N418; eBioscience), anti-Gr1 (Ly-6G and Ly-6C)-APC (clone RB6-8C5; BD Biosciences), anti-Siglec F-PE (clone E50-2440; BD Biosciences) and anti-MHCII (I-A/I-E)-PE-CY-7 (clone M5/114.15.2; eBioscience). Cells were incubated with antibodies at 4°C for 30 min while protected from light, then pelleted and resuspended in PBS with 0.5% BSA at 10⁸ cells/mL. Samples were evaluated and sorted using a FACSAria Fusion cytometer (BD Biosciences). Eosinophils were identified as CD45⁺/CD11c^-/GR1^lo/MHCII^-/Siglec F⁺ cells. Additional details are provided in S1 Appendix.