100 m cell strainer

Primary EqS and ctrl Fbs were established from tissue samples excised from the core of the tumor or the dermis, respectively. Each tissue sample was minced (1 mm³ pieces), washed in PBS, and frozen at −150 °C in 50% DMEM (high glucose, L-glutamine; Gibco/Thermo Scientific, Waltham, MA, USA), 40% FCS (Gibco/Thermo Scientific) and 10% DMSO (Merck Millipore, Darmstadt, Germany). For the setup of cell cultures, tissue samples were thawed, washed twice in DMEM, and digested at 37 °C for 60 to 80 min in DMEM containing collagenase IV (2 mg/mL, MilliporeSigma, Darmstadt, Germany). After digestion, the tissue was mechanically dissociated using four flame-constricted Pasteur pipettes with decreasing luminal diameter, filtered (100 µm cell strainer; Thermo Fisher Scientific, Waltham, MA, USA) and washed twice in DMEM with 10% FCS and 1% penicillin-streptomycin (Gibco/Thermo Scientific). Cells were cultured in T150 cell culture flasks (TPP, Trasadingen, Switzerland) at 37 °C and 5% CO₂. For trypsinization, 0.25% trypsin (Gibco/Thermo Scientific) was used. All primary cultures were tested negative for mycoplasma using the PlasmoTestTM—Mycoplasma Detection (InvivoGen, Toulouse, France).

Following euthanasia, newborn (PND0) mouse lungs were perfused intracardially through the right ventricle with cold PBS. Lung tissue was minced and enzymatically digested in RPMI-1640 (Corning 10-040-CV) containing 50 U/ml DNase I (Roche, 10104159001), 400 µg/ml Liberase TM (Roche, 5401119001) at 37°C for 15 min. Tissue homogenates were passed through a 100 µm cell strainer (Thermo Fisher Scientific), followed by erythrocyte lysis using ACK buffer (Life Technologies). Before antibody staining, the cell suspensions were filtered through a 70 µm cell strainer (Thermo Fisher Scientific). Single cell suspensions were resuspended in PBS with CD16/CD32 blocking antibody (BioLegend, 101320) and Zombie Aqua (BioLegend, 423102) for 20 min. The cells were then incubated with fluorochrome-conjugated antibodies against CD45, CD31 and EPCAM (BioLegend, 109828, 102507 and 118215, respectively) in staining buffer (HBSS containing 2% FBS and 2 mM EDTA) for 1 h. Samples were analyzed on a NovoCyte Penteon Flow Cytometer (Agilent). Data were analyzed using FlowJo software.

Serum was isolated by centrifugation of SST vacutainers at 1,300×g for 10 minutes (min) at room temperature and stored at −80°C. PBMC were isolated from heparinized blood diluted 1:1 in DPBS by centrifugation in a Leucosep tube (Thermo Fisher Scientific) containing Histopaque 1.077 (Merck Life Science). After centrifugation, the plasma was removed and PBMC were harvested and washed in DPBS. Contaminating erythrocytes were lysed by incubation in RBC Lysis Buffer (BioLegend, London, UK) for 5–7 min. PBMCs were washed twice with DPBS and resuspended in cRPMI for immediate use or cryopreserved in 10% DMSO (Merck Life Science) in FBS. BALF was centrifuged at 500×g for 10 min, and the supernatant was aliquoted and stored frozen at −80°C. The BALC were washed twice in DPBS and filtered through a 100 µm cell strainer (Thermo Fisher Scientific). The spleen, thymus, and inguinal lymph node tissues were finely chopped in DPBS and the cells were dissociated by applying pressure to the syringe barrel. Cells were then passed through a 100 µm cell strainer and washed, and erythrocytes were lysed before two final washes in DPBS. BALC and lymphoid tissue cells were resuspended in cRPMI or cryopreserved as described for PBMC.