Zombie nirtm fixable viability dye

Eosinophil cytotoxicity assays were carried out as described before.^{7 (link)} IL-5 Eos (or IL-33 Eos) were co-incubated with CT26 cells (4x10⁴) in a 96-well plate at different ratios for eosinophils to tumor cells (E:T). To be able to differentiate between eosinophils and CT26 cells, we either used GFP⁺ eosinophils from CByJ.B6-Tg(UBC-GFP)30Scha/J mice (Jackson Laboratory) or we stained CT26 cells with eFluor^TM 450 (500 nM). After 6, 7 or 24 hrs, eosinophils were collected, and CT26 tumor cells were detached using trypsin/EDTA (PAN Biotech; P10-023100). Cells were stained with Annexin-V (BD Biosciences; # 5566547; according to the manufacturer’s protocol) or Zombie NIR^TM Fixable viability dye (Biolegend; # 423105). The percentage of dead CT26 cells (or living GFP⁺ eosinophils) was then analyzed as Annexin-V or Zombie NIR^TM Fixable viability dye positive or negative cells using flow cytometry.

Flow cytometry was performed on a CytoFLEX (Beckman Coulter, Brea, CA, United States) flow cytometer. Antibodies for flow cytometry were titrated over a range of concentrations prior to use. The following controls were included in all flow cytometry runs: unstained control, fluorescence-minus-one (FMO) controls, secondary-only controls, and single-color compensation controls for fluorochromes. UltraComp compensation beads (Cat# 01-2222-43, ThermoFisher) were used with antibodies raised in mice.
Live single-cell suspensions were labelled with Zombie NIR^TM fixable viability dye (Cat# 423105, BioLegend^®, San Diego, CA, United States) to eliminate dead cells during the analysis stage. Next, the cells were fixed in 10% buffered formalin for 10 min at 4°C in the dark. After fixation, the cells were permeabilized (Ca²⁺Mg²⁺-free PBS with 0.5% BSA supplemented with 0.1% Triton X-100) for 15 min at room temperature in the dark. Primary and secondary antibodies (used at dilutions according to Table 2) were incubated for 30 min at 4°C in the dark. Flow cytometric analysis was performed using FlowJo software (version 10.7.1).