Facsmelody flow cytometer

After the 22-h seeding periods, supernatants were recovered and centrifuged at 300×g for 10 min for each seeding techniques. Cell-seeded PETG rods were incubated with trypsin 0.05% (Fisher Scientific)/EDTA 0.01% (Teknisciences) for 10 min to let the cells detach, and then centrifuged at 300×g for 10 min. The recovered cells from supernatant and scaffolds were incubated for 15 min with Calcein AM 2.5 nM (Thermo-Fisher) to label live cells and Ethidium homodimer-1 4 μM (Thermo-Fisher) to label dead. Cells were immediately analysed with a BD FACSMelody™ flow cytometer (BD Biosciences) and data were treated using the FlowJo™ v9 software (Ashland, OR, USA). For mature tissues, they were first recovered from the scaffolds and placed in a digestion solution of 5.7U/ml collagenase H (Sigma-Aldrich) in accutase (Sigma-Aldrich) at 37 °C with 300 rpm agitation for 30 min. Cells were filtered through a 40 μm cell strainer (Fisher Scientific) and centrifuged at 300×g for 10 min. Finally, cells were analysed as described below.

Flow cytometry was performed using a BD FACSMelody flow cytometer (BD Biosciences, USA) equipped with two lasers (488 nm and 635 nm) and a Gallios flow cytometer (Beckman Coulter, USA) equipped with three lasers (488 nm, 635 nm, and 405 nm). The data were analysed using FlowJo software v10.6.1 (BD Biosciences, USA).

ROS were detected in human NSCLC lines using the cell permeable fluorophore CellROX Green (ThermoFisher Scientific). Cells were seeded in 6-well plates densities stated above in 2 mL media 24 h prior to performing the assay. A final concentration of 1 μmol/L CellROX Green reagent was added to each well and incubated for 30 min at 37 °C, protected from light. Cells were then washed with PBS, harvested using 0.05% trypsin-EDTA and suspended in 1 mL of ice-cold Hanks balanced salt solution. The cell suspension was passed through a 35 μm filter and kept on ice prior to analysis on BD FACS Melody flow cytometer (488 nm laser and 527/32 bandpass filter; BD Biosciences). 20,000 single cell events were recorded per sample with the data gated post-acquisition based on forward (FS) and side scatter (SS) profiles to include only single cell events and to exclude cellular debris.

The differentiation of T cells was performed using standard methods as described before (24 (link)). Briefly, C57BL/6 mice were injected i.v. with 20 µg LPS. After 48 h, aortic single cells and splenocytes were isolated and stimulated with cell-stimulation cocktail plus protein transport inhibitors (00-4975-93, Invitrogen, San Diego, CA, USA) for 4–6 h at 37°C under 5% CO₂ environment. After that, cells were washed and then stained with CD45, CD11b, CD19, CD3, CD4, and CD8. Cells were then fixed, perfornated, and stained intracellularly with IFN-γ, IL-17A, IL-10, and TGF-β1 or cells were fixed, perforated by the Foxp3 buffer set (560409, BD, San Diego, CA, USA), and then stained intracellularly with Foxp3 (560403, BD). Flow-cytometric analysis was performed with BD FACS Melody flow cytometer.

The viability of isolated uNK cells was determined by incubation with Fixable Viability Stain 660 (FVS660, BD Biosciences, NJ, USA) diluted 1:1000 in PBS for 10 minutes at 4°C. For analysis of purity, cell suspensions were incubated with CD45‐FITC (555482, 1:5, BD Bioscience) and CD56‐PE (555516, 1:5, BD Bioscience) for 30 minutes in the dark at 4°C. After two washes with FACS buffer (0.5% BSA and 2 mM EDTA in PBS), cells were resuspended in 500 µL of FACS buffer prior to analysis on BD FACSMelody flow cytometer.

Cytokines of vascular DCs were estimated by intracellular staining as described previously (23 (link)). Briefly, C57BL/6 mice were injected i.v. with 5 µg lipopolysaccharide (LPS) (L4391, Sigma, St. Louis, MO, USA). After 12 h, aortic single cells and splenocytes were isolated from mice and stimulated with GolgiSTOP Protein Transport Inhibitor (51-2092KZ, BD) for 4–6 h at 37°C under 5% CO₂ environment. Cells were washed and then stained with antibodies against CD45, CD64, CD11c, CD11b, MHCII, B220, and XCR1. Cells were fixed, perforated, and stained intracellularly with IL-12/IL-23 p40, IL-1β, and IL-10 for 30 min on ice. Flow-cytometric analysis was performed with BD FACS Melody flow cytometer.