Aurora analyzer

PBMCs were quickly thawed at 37 °C. 200 µL of the suspension, consisting of cells and freezing media, were washed once with PBS 1X, stained with the eleven fluorochrome conjugate antibodies specified in Supplementary Table S6, incubated for 30 min at +4 °C and analyzed by the Cytek Aurora analyzer (Cytek Biosciences, Fremont, CA, USA).
Briefly, cell viability was measured by using 7-aminoactinomycin D (7-AAD), an intercalating dye that can pass through the compromised cell membrane and bind the DNA, thus live cells will be 7-AAD negative.
B cells were characterized for their maturation stages using IgDvsCD27 and IgDvsCD38 markers; the expression of immunoglobulin A and D were also assessed [16 (link)].
Helper (CD4+) and cytotoxic-enriched T cells (CD8e) were characterized for their maturation stages by the expression of the phosphatase isoform CD45RA and the chemokine receptor CCR7 [17 (link),18 (link)]. See Figure 1 for details.
Overall, lymphocyte viability and 27 cell frequencies with respect to hierarchically higher cells population were assessed.
The remaining cell suspension was frozen by using the Mr Frosty container to achieve the temperature of −80 °C, then transferred at −150 °C. Thawing and freezing procedures was repeated twice for each sample.
Fresh sample characterization and cell frequencies are from Orrù et al., 2020 [1 (link)].

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.