Hepes buffer

PBMC were isolated by Pancoll gradient centrifugation (PAN-Biotech, Aidenbach, Germany). Monocytes were isolated by positive selection with anti-CD14 microbeads. The purity was analyzed by flow cytometry on a FACS LSRII SORP (BD Biosciences, Heidelberg, Germany) with anti-human CD14 and ranged from 90%-99% (S8B Fig). Remaining PBMC were frozen in RPMI 1640 supplemented with 20% FCS and 20% DMSO (both from Sigma-Aldrich, Munich, Germany) for subsequent isolation of autologous T cells.
Isolated monocytes were counted using trypan blue exclusion (Applichem Panreac, Darmstadt, Germany) and seeded at a density of 1.5*10⁶ cells/ml in RPMI 1640 (Gibco, Life science, Darmstadt, Germany), supplemented with 10% FCS (Sigma-Aldrich Chemie GmbH, Munich, Germany), 1% penicillin/streptomycin, 1% L-Glutamine and 1% HEPES buffer (all from Biochrom, Berlin, Germany), 50 μM 2-Mercaptoethanol (Sigma-Aldrich, Munich, Germany), 50 ng/ml human GM-CSF and 20 ng/ml human lL-4. Cells were incubated at 37°C and 5% CO₂, medium exchanged on after 3 days and cells harvested on day 6. Successful differentiation of monocytes into monocyte-derived dendritic cells (MoDC) (CD14^-, HLA-DR^high, CD11c⁺, CD83^dim) was confirmed by flow cytometric analysis (S8A Fig).

BMDM were isolated from WT mice by flushing a femoral bone with 10 ml of sterile VLE RPMI supplemented with 1% penicillin–streptomycin using a sterile cannula. The obtained cell suspension was centrifuged at 500×g for 5 min at 4 °C and resuspended in macrophage differentiating medium containing 10 ml VLE RPMI supplemented with 1% non-essential amino acids (NEA; 100×; Biochrom AG, Berlin, Germany), 1% HEPES-Buffer (1 M; Biochrom AG), 1% sodium pyruvate (100 mM; Biochrom AG), 10% L929-CM, 10% FBS and 1% penicillin–streptomycin. 5 × 10⁵ cells were seeded into each well of a 24-well plate. After 72 h, 100 µl of differentiating medium was added to each well. Cells were allowed to differentiate for 6 days (Naujoks et al. 2016 (link)). Identically to alveolar macrophages, medium was replaced by 400 µl of complete medium 24 h prior to stimulation.

RAW 264.7 (ATCC TIB-71) represents a murine (BALB/c; H2^d) adherent growing monocyte/macrophage cell line which is transformed by Abelson murine leukemia virus [25 (link)]. RAW 264.7 cells are capable of pinocytosis and phagocytosis, antibody-dependent lysis of tumor cells as well as nitric oxide (NO) and cytokine production and they are responsive to LPS [25 (link)]. Cells were cultured in 175 cm² cell culture flasks (Greiner Bio-One, Frickenhausen, Germany) in phenol-red free RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES buffer, 100 μg/mL penicillin/streptomycin (Biochrom, Berlin, Germany), and 50 μM β-mercaptoethanol (Sigma Aldrich, Steinheim, Germany) at 37°C, 5% CO₂, and 95% air humidity.
For experiments the cells were activated with heat-killed Salmonella enterica Serovar Enteritidis (SalmoVac SE, IDT Biologika GmbH, Dessau-Rosslau, Germany). The relative antigen concentration used in this study (ratio: 10⁸ hk S.E. to 10⁷ macrophages) was previously determined (data not shown) and ensures appropriate activation of macrophages.

Within this study we worked with several established human squamous cell carcinoma (SCC) lines of the head and neck. The FaDu_DD [37 (link)] cells originate from the hypopharynx; SAS (JCRB Cell Bank, NIBIOHN, Osaka, Japan), Cal33 (DSMZ, Braunschweig, Germany) and UT-SCC-5 cells (DSMZ) from the tongue. The metastatic cell line Detroit562 (CLS, Eppelheim, Germany) derives from pleural effusion of a primary tumor of the pharynx. The keratinocyte cell line HaCaT (DKFZ, Heidelberg, Germany) was used as normal tissue control. These established HNSCC cell lines were cultivated as monolayer with Dulbecco’s modified Eagle medium (DMEM; Biochrom, Cambridge, UK) containing 10% fetal calf serum (FCS, Sigma-Aldrich, St. Louis, MO, USA), 2% HEPES buffer (1 M, Biochrom), 1% non-essential amino acids (NEA 100×, Biochrom), 1% sodium pyruvate (Biochrom), and 1% penicillin/streptomycin (10,000 U/mL, Biochrom). The incubator was adjusted to 37 °C and 5% CO₂. Cells were passaged usually twice per week after a confluency of 70–80% was reached. Cells were only used for experiments until passage 15 and regularly tested for cell authentication and mycoplasma infection. Irradiated (IR) sublines were generated from indicated parental cell line by selection with at least 15 fractions of 4 Gy and analyzed together with age-matched controls [36 (link)].

Islets were isolated as described before by our department [39 (link)]. Briefly, the extracted pancreas was perfused with 4 mg / ml collagenase B (Roche, Mannheim, Germany) dissolved in 1% Hank’s solution (Biochrom, Berlin, Germany) supplemented with 35 ml Hepes buffer (Biochrom, Berlin, Germany), 10 ml Ciprofloxacin, 10 ml Penicillin-Streptomycin, and 1 ml Gentamycin through the ductus pancreaticus. The perfused pancreas was mechanically chopped with scissors before 10 minutes of incubation in collagenase solution at 37°C in a shaking water bath. After every 3 minutes of collagenase digestion the sample was vortexed for 10 seconds. The digested tissue was shaken by hand for two more minutes and the digestion process was eventually stopped by placing the tube containing the tissue on ice and adding cold Hank’s solution. Following 3 minutes of centrifugation at 1500 rpm, the supernatant was discarded and the pellet was dissolved in 15% P/FCS dissolved in Medium 199 (Gibco, Karlsruhe, Germany) and fetal bovine serum (biowest, Nuaillé, France) at room temperature. The islets were hand-picked under stereomicroscope and incubated overnight at 37°C to overcome the isolation stress.

Cells were seeded in duplicates with a density of 2 × 10⁴ cells in 50 µL per well on an 8-well Nunc™ Lab-Tek™ Chambered Coverglass dish (Thermo Fisher Scientific, Rochester, NY, USA) and incubated for 24 h together with the compounds. Immediately before analysis, 5 µL of 20 µM HEPES buffer (Biochrom GmbH, Berlin, Germany) was added to each cavity. Subsequently, 2 µL of reduced MitoTracker^®Red-H₂XROS (Invitrogen by Thermo Fisher Scientific, Eugene, OR, USA) and 2 µL of Wheat Germ Agglutinin (WGA)-Alexa Fluor™ 488 conjugate (Invitrogen by Thermo Fisher Scientific, Eugene, OR, USA) were added and incubated for 15 min. Hoechst 33342 (Invitrogen by Thermo Fisher Scientific, Eugene, OR, USA) was added 5 min before the cells were analyzed by confocal microscopy utilizing an inverted microscope (Zeiss Axio Observer Z1, Zeiss, Oberkochen, Germany) in arrangement with a spinning disk confocal system (UltraVIEW VoX, PerkinElmer, Waltham, MA, USA). All the images were generated using a 40× water immersion objective (Zeiss, Vienna, Austria).