Live dead fixable near ir or blue dead cell staining kit

5 × 10⁴ WT or Enpp1^−/− 4T1-luc cells were orthotopically injected into WT or Enpp1^−/− BALB/cJ mice respectively. Starting the next day, mice were intratumorally injected with 100 μL of 100 μM neutralizing (WT) or non-binding (R237A) STING every other day up to day 13. Mice were killed on day 15 and tumors were collected and digested in Roswell Park Memorial Institute (PRMI) + 10% FBS with 20 μg/ml DNase I type IV (Sigma-Aldrich) and 1 mg/mL Collagenase from Clostridium histolyticum (Sigma-Aldrich) at 37 °C for 30 min. Tumors were passed through a 100 μm cell strainer (Fisher Scientific) and red blood cells were lysed using red blood cell lysis buffer (155 mM NH₄Cl, 12 mM NaHCO₃, and 0.1 mM Ethylenediaminetetraacetic acid) for 5 min at room temperature. Cells were stained with Live/Dead fixable near-IR or blue dead cell staining kit (ThermoFisher). Samples were then fixed and permeabilized with eBioscience FOXP3/Transcription Factor Staining Buffer Set (Invitrogen), Fc-blocked for 10 min using TruStain fcX (BioLegend) and subsequently antibody-stained with antibodies. Cells were analyzed using a Symphony (BD Biosciences), or an Aurora analyzer (Cytek). Data was analyzed using FlowJo V10 software (BD) and Prism 9.1.0 software (GraphPad) for statistical analysis and statistical significance was assessed using the unpaired t test with Welch’s correction.