Zombie violet dye

Cell division was analyzed by the fluorescent CytoTell Green dye (AAT Bioquest, Sunnyvale, CA, USA) according to the manufacturer’s guidelines. Briefly, 3 × 10⁵ cells were centrifuged and stained with 2 µL of CytoTell dye diluted in 1 mL of serum-free DMEM and incubated at 37 °C for 30 min. After centrifugation, half of the cells (1.5 × 10⁵) were plated into T25 cell culture flasks in 5 mL of media and analyzed at day 5. Day 1 samples were co-stained with Zombie Violet dye (423113, Biolegend) to exclude dead cells and analyzed by Attune™ NxT flow cytometry.

Bronchoalveolar lavage was performed by first exposing the trachea of euthanized mice.

The exposed trachea was punctured using Vannas Micro Scissors (VWR), and 1 ml PBS was injected using a 20G‐1” IV catheter (McKesson) connected to a 1‐ml syringe.

The PBS was flushed into the lung and aspirated three times, and the recovered fluid was placed in a 15‐ml tube on ice.

The 1 ml PBS wash was then repeated three additional times for a total of 4 ml recovered fluid.

Cells were filtered, spun down, and resuspended in a 96‐well plate for antibody staining.

Cells were suspended in 1X PBS (pH 7.4) containing 0.01% NaN₃ and 1% fetal bovine serum (i.e., FACS buffer).

Fc receptors were blocked with anti‐CD16/32 (2.4G2, BD Pharmingen). Cell viability was assessed using Zombie Violet dye (BioLegend). Surface staining included antibodies specific for murine Siglec F (E50‐2440, BD Pharmingen), CD11b (M1/70, BioLegend), CD64 (X54‐5/7.1, BioLegend), CD45 (104, BioLegend), CD3 (17A2, eBiosciences), and CD19 (1D3, eBiosciences). Cell sorting was performed on a FACSAria (BD Biosciences). Cells were collected in complete media, spun down, resuspended in TRIzol, and frozen at −80° overnight prior to RNA isolation.

PMNs were stimulated with SKMEL28 CM, A375 CM, HEMa CM or control medium for 90 min (37 °C, 5% CO₂). To determine the viability of the cells (2.5 × 10⁵), Zombie Violet dye (BioLegend, San Diego, CA, USA) was added. The cells were then stained with phosphate buffered saline (PBS) containing 1% FCS for 20 minutes at + 4 °C. Allophycocyanin (APC)-conjugated anti-CD66b (clone REA306, 1:50), VioBlue-conjugated anti-CD193 (clone REA574, 1:10), PerCP-conjugated anti-CD11b (clone REA713, dilution 1:50) and FITC-conjugated anti-CD62L (clone 145/15, dilution 1:50) were used, all from Miltenyi Biotech (Bergisch Gladbach, Germany). The MACS Quant Analyzer 10 and FlowJo software version 10 were used to analyze the results. Doublets and debris (identified based on forward and side scatter properties), dead cells (identified using the Zombie Violet Fixable Viability Kit) and eosinophils (identified based on the CCR3⁺ exclusion gate) were excluded from the analysis. All the experiments were run in duplicate.

After treatment, cells were trypsinized, washed once in PBS, and resuspended in Blocking Buffer (2% FBS, 0.5% BSA in PBS) for 15 min on ice under agitation. Cells were stained for 30 min at +4 °C with APC or PE-conjugated anti-CD133 antibodies (diluted at 1/200 or 1/400 respectively; Biolegend, San Diego, USA) or corresponding control Immunoglobulin G1 (IgG1, Biolegend) antibody as a control for non-specific staining. After washes, pellets were resuspended in PBS with MitoTracker^TM Deep Red FM (MT) (m22426, Life Technologies), MitoStatus TMRE (564696, BD Biosciences, San Jose, CA, USA) or Cell ROX Deep Red reagent (1691766, Life Technologies) for 20 min at room temperature. Annexin V-APC staining was performed on attached and floating cells according to manufacturer’s instructions (550474 & 556454, BD Biosciences, San Diego, CA, USA). Zombie Violet Dye (77477, Biolegend, San Diego, CA, USA) was used to exclude non-viable cells. 50,000 cells per sample were analyzed using a FACS Canto II (BD, Franklin Lakes, NJ, USA) and analyzed with FlowJo 9.2 software (Ashland, OR, USA). Moreover, viable cells corresponding to the CD133 negative and positive populations were sorted into 5 mL tubes containing full RPMI medium using a SONY SH800S instrument (SONY, Tokyo, Japan).