Red blood cell lysis

On days 1 through 4 after transplant, mice were sacrificed. Mice were first perfused with 10mL basic salt solution (BSS) through their native heart, followed by an additional 10mL BSS perfusion of the transplanted heart. The transplanted heart was then excised, and both left and right ventricles were cut open to assess for major clots. Identified clots were then removed from the chambers as necessary, and the sample was then placed in BSS on ice. A single cell suspension was obtained from a modified cold-active protease tissue digestion protocol as previously described (61 (link), 62 (link)). Debris from the sample was removed (Miltenyi Biotec), followed by red blood cell lysis (eBioscience). Cells were then counted and plated to prepare for staining.

Cells were generated from naïve mice as previously described (28 (link)). Briefly, bone marrow was removed from femurs and tibiae. After red blood cell lysis (eBioScience, San Diego, CA, USA), the total bone marrow cells (2.5 × 10⁵ cells/ml) were cultured in RPMI 1640 supplemented with 5% heat-inactivated FBS, 10 mM HEPES, 20 µg/ml gentamycin, 100 U/ml penicillin–streptomycin, 2 mM l glutamine and 50 µM 2-ME (Gibco, Invitrogen, Burlington, ON, Canada). Complete medium was complemented with 20% GM-CSF from a mouse GM-CSF-transfected cell line (Ag8653) as a source of GM-CSF (35 (link)). Cells were cultured for 7 days at 37°C in a 5% CO₂ incubator, and fresh medium was added on days 3 and 5. On day 7, clusters were harvested and subcultured overnight to remove adherent cells. Non-adherent cells were harvested on day 8, washed, and used as immature DCs for the studies in complete medium containing 5% GM-CSF. Cell purity routinely comprised 86–90% CD11c^high, as determined by FACS analysis and as previously reported (28 (link)).

To detect intracellular cytokines and chemokines by flow cytometry, mice administered with Proleukin^®/IL-2 and saline-treated control groups of the same experiment were injected with 0.25 mg of BFA (Sigma-Aldrich, USA) at the endpoint of the experiment (144 h) and sacrificed 6 h later. Submandibular blood collection was carried out in EDTA tubes (Greiner Bio-One, Austria), and red blood cells (RBCs) were lysed using RBC lysis buffer (Life Technologies, USA) prior to flow cytometry staining. Spleen and lymph nodes were digested in a mixture of collagenase IV (GIBCO, UK), DNase I (Sigma Aldrich, USA) and meshed through a 70 µm filter (Thermo Fisher Scientific, USA) in DMEM medium (Thermo Fisher Scientific, USA). When necessary, cell suspensions were subjected to red blood cell lysis (GIBCO, UK). The single-cell suspension was washed and re-suspended in media supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, USA).

Freshly resected lung tissue samples were cut into small pieces and digested for 1 h at 37 °C using the tumor dissociation kit (Milteniy Biotech, Bergisch Gladbach, Germany) and the gentleMACS Dissociator (Milteniy Biotech). Digested samples were filtered through a 100 µm cell strainer, washed, and red blood cell lysis (Gibco, Waltham, MA, USA) was performed. CD45⁺ immune cells were positively sorted using CD45 (TIL) microbeads (Milteniy Biotech) according to manufacturer recommendations. Recovered cells were stained for 30 min with fluorochrome-labelled primary antibodies in PBS 1% HS, 1% FCS at 4 °C. Cell viability was ascertained by labeling with fixable viability dyes (eBioscience, San Diego, CA, USA). For intracellular staining, cells were washed in PBS and next fixed in PFA 4% for 10 min at room temperature and permeabilized with PBS 1%, FCS 1%, HS 0.1% saponin (permeabilization buffer) for 10 min. Cells were next incubated with the following antibodies VEGF-PE (R&D systems) and OPN-FITC (R&D systems, Minneapolis, MI, USA) in permeabilization buffer for 45 min at RT. Cells were next washed in PBS and proceeded to flow cytometry analysis. Stained cells were acquired using a Fortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with FlowJo V10.4.2 software (TreeStar, Antwerp, Belgium).