Accutase solution

Microglia was treated for 15 m with H₂DCFDA (DCF, Invitrogen) 1:5000 diluted in PBS. Then cells were detached using Accutase Solution (Merck) 10 m at 37 °C. Data were acquired using a FACSCantoII (BD Bioscience) and were analyzed using FCS Express 7 (De Novo Software). Unstained samples were employed to set gates.

To profile 3D-UHU cluster of differentiation (CD) cell surface markers, terminally differentiated cultures were dissociated using Accutase solution (Merck, UK). Cells were washed with PBS and filtered (100 µm) to prepare single cell suspensions. BD Horizon Fixable Viability Stain 780 solution (1:1000) was added to the cell suspension and incubated at room temperature (RT) for 15 min. Cells were washed and blocked with blocking buffer plus Fc receptor blocking antibody (Clone Fc1.3216, BD Biosciences, UK). Cells were incubated at RT for 10 min then centrifuged (800 g, 3 min). BD Horizon Brilliant Stain Buffer was added to each sample followed by the following antibodies at the optimised volume per test (Supplementary Table 1): CD9 (BV421, Clone M-L13), CD44 (Alexa Fluor 700, Clone G44-26), CD47 (BV786, Clone B6H12), CD49b (Alexa Fluor 647, Clone AK-7), CD59 (BUV 395, Clone p282), CD63 (PE-Cy7, Clone H5C6), CD95 (BUV737, Clone DX2), CD104 (BV480, Clone 439-9B), CD227 (BV650, Clone HMPV), CD271 (BV711, Clone L128). Cells were incubated at 4°C in the dark for 30 min then washed and resuspended in wash buffer with 1% formaldehyde solution. Data acquisition was performed using Cytek Aurora equipped with 5 lasers. The flow cytometry results were analysed using FlowJo™ v10.8 Software (BD Life Sciences).

3T3-L1 preadipocytes were sponsored by the Department of Nutritional science Vienna. Cells were cultured as a monolayer in Dulbecco's modified Eagle medium (DMEM) high glucose (4.5 g/l) containing L-glutamine, 5% penicillin/streptomycin and 10% fetal calf serum at 37°C in a humidified atmosphere of 95% air and 5% CO2. Studies were performed in the passage numbers 3 to 5. Cells were passaged before reaching confluency using 1x PBS and Accutase solution (all substances from Merck, Germany). This cell line was chosen because senescence in preadipocytes impairs their function and the cytokines released in senescent cells can be spread to nonsenescent neighbored preadipocytes. Moreover, the release of cytokines is highly secreted in preadipocytes of older rats and can impair the recruitment of immune cells [15 (link)].

The human head and neck cancer cell lines A‐253 (RRID: CVCL_1060), Detroit 562 (D‐562, RRID: CVCL_1171), FaDu (RRID: CVCL_1218), SCC‐9 (RRID: CVCL_1685), and SCC‐15 (RRID: CVCL_1681) were ordered from ATCC (Manassas, VA, USA) as Head and Neck Panel TCP‐1012. The head and neck cancer cell line PCI 52 (RRID: CVCL_RJ99) was kindly provided by Prof. Dr. Theresa. L. Whiteside [University of Pittsburgh Cancer Institute (PCI), Pittsburgh, PA, USA] in 2013 [30 (link)]. HNC cell lines were maintained in DMEM (PanBiotech, Aidenbach, Germany) supplemented with 10% fetal calf serum (FCS, Gibco, Carlsbad, CA, USA), 1% l‐glutamine (Sigma‐Aldrich, St. Louis, MO, USA) and 1% penicillin/streptomycin (Sigma‐Aldrich) at 37 °C in a 5% CO₂ humidified atmosphere. The medium was changed every 2–3 days, and the cells were passaged prior reaching confluence. Detachment of adherent cells was achieved by incubation with 0.05% Accutase solution (Merck, Darmstadt, Germany) for 5–10 min at 37 °C. Patient samples were provided from HNSCC patients from the Department of Oral and Maxillofacial surgery, University Hospital Regensburg, and processed immediately after resection. All cell lines are regularly tested for mycoplasma. The used cell lines were free of mycoplasma.

Human monocytes, macrophages and dendritic cells were cultured in RPMI1640‐Glutamax medium supplemented with 1% penicillin/streptomycin 10% FCS at 37°C in 5% CO₂ atmosphere. Buffy coats from healthy donors were obtained freshly from DRK‐Blutspendedienst. Orangu assay was purchased from Cell guidance systems. Human FcR Blocking Reagent, human CD14 MicroBeads, human granulocyte macrophage colony‐stimulating factor (GM‐CSF), macrophage colony‐stimulating factor (M‐CSF), bovine serum albumin (BSA), IL‐4 and all antibodies for surface staining were purchased from Miltenyi Biotec. Cytometric bead array was purchased from BD Biosciences. ELISA for CCL18 and CCL17 was purchased from BosterBio and BioLegend, respectively. Accutase^® solution and Biocoll were purchased from Merck. EDTA was purchased from Sigma‐Aldrich. Silvestrol (purchased from the Sarawak Biodiversity Centre, Kuching, Borneo at a purity of >99%) was dissolved in DMSO and further diluted in medium (c_stock = 6 mmol/L, maximal DMSO concentration during experiments 0.000083% v/v).

Immortalised human brain microvascular endothelial cells (I-HBMEC) (Innoprot, Bizkaia, Spain) (P10361-1M), Immortalised human astrocytes (IA) (Innoprot, Bizkaia, Spain) (P10251-1M), CLTH/Immortalised Pericytes (IP) (Amsbio, Oxfordshire, U.K.) (CL05008-CLTH), D-MEM/F:12 (1:1) (CE) (Thermofisher, Cambridge, U.K.) (11320074), Insulin-trans-sel-G, 100× (Thermofisher, Cambridge, U.K.) (41400045), Penicillin streptomycin (PS) (Gibco, Paisley, U.K.) (15070-063), Fetal bovine serum (FBS) (Gibco, Paisley, U.K.) (A3160801), Hydrocortisone solution (50 um) (Merck, Dorset, U.K.) (H6909), Fibroblast growth factor-basic (BFGF) (Merck, Dorset, U.K.) (SRP3043), Heparin sodium salt (Merck, Dorset, U.K.) (H3149), Dulbecco’s phosphate buffered saline, M (DPBS) (Merck, Dorset, U.K.) (D8537), Accutase^® solution (Merck, Dorset, U.K.) (A6964), Tissue culture flask, 75 cm² growth area (T75) (Sarstedt, Leicestershire, U.K.) (83.3911.002).