Propidium iodide

Growth medium RPMI 1640, trypsin/EDTA and fetal bovine serum were purchased from Cytogen (Zgierz, Poland). A growth medium M-254 and human melanocytes growth suplement-2 (HMGS-2) were acquired from Cascade Biologics (Portland, OR, USA). NC-Slides (A2 and A8), Via-1-Cassette (containing acridine orange and DAPI), Solution 3 (1 μg/mL DAPI, 0.1% triton X-100), Solution 5 (400 μg/mL VitaBright-48, 500 μg/mL propidium iodide, 1.2 μg/mL acridine orange), Solution 7 (200 μg/mL JC-1), Solution 8 (1 μg/mL DAPI), Solution 15 (500 μg/mL Hoechst 33342), and Solution 16 (500 μg/mL propidium iodide) were obtained from ChemoMetec (Lillerød, Denmark). Annexin V-CF488A conjugate and Annexin V binding buffer were obtained from Biotium, Inc. (Fremont, CA, USA). Ketoprofen—KTP (chemical name: (RS)-2-(3-benzoylphenyl)-propionic acid), amphotericin B, penicillin, and dimethyl sulphoxide (DMSO) were obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA). Other chemicals were from POCH S.A. (Gliwice, Poland).

Image cytometry with an NC-3000 Image Cytometer (ChemoMetec, Allerod, Denmark) was used for cellular and mitochondrial phenotyping. A minimum number of 5000 events were analyzed in each experiment. The following assays were executed according to the manufacturer’s recommendations: cell count (acridine orange and DAPI (ChemoMetec)), mitochondrial membrane potential (JC-1 and DAPI (ChemoMetec)), thiol redox status (VitaBright-48, Propidium iodide, and acridine orange (ChemoMetec)). Mitochondrial superoxide levels (MitoSOX™ (Thermo Scientific) and Hoechst-33342 (ChemoMetec)) were measured as described earlier [43 (link)]. To measure mitochondrial mass, 100 nM fluorescent dye MitoTracker™ Green FM (Invitrogen, Carlsbad, CA, USA) diluted in Hank’s balanced salt solution (HBSS) (Invitrogen) was added to cells for incubation for 30 min at 37 °C. After removing excess dye, the cells were collected and a 1:1000 dilution of RedDot2 (Biotium, Fremont, CA, USA) (concentration not specified) was added to each sample before measurement. The NucleoView NC-3000 software (ChemoMetec) was used for data analysis.

Donor pancreata were processed by the Beta Cell Bank of the JDRF Centre for Beta Cell Therapy in Diabetes (Brussels, Belgium) affiliated to the Eurotransplant Foundation (Leiden, The Netherlands). Full written consent for use of donor material for research was obtained according to Belgian laws. This project was approved by the Medical Ethical Committee of the Hospital of the Vrije Universiteit Brussel. Isolation of the exocrine cell fraction was performed as previously described^{14 (link)}. Advanced RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA; 12633020) containing 5% foetal bovine serum (Biochrom GmbH, Berlin, Germany; S0115) and 1% penicillin-streptomycin solution (Merck, Darmstadt, Germany; P0781) was used for culture of the exocrine cells under 5% CO2 atmosphere at 37 °C. Culture medium was renewed on day 1, day 2 and day 4. TGF-beta inhibitor Alk5iII (Enzo LifeSciences; Farmingdale, New York; Alx-270-445) was added at a concentration of 10 µM from start of culture. Cells (100 µl cell pellet per 10 ml medium) were cultured in suspension in 94/16 mm petri dishes (Greiner Bio-one; Kremsmünster, Upper Austria; 633180). Cell loss in culture was analysed by counting the number of nuclei after cell lysis (ChemoMetec, Denmark) and propidium iodide (Sigma) staining.

Cortical organoids, primary fetal neurons, and astrocytes were manually dissociated and resuspended in Annexin V binding buffer (Invitrogen). Next, Annexin V-CF488A conjugate (Biotium, Inc., Hayward, CA, USA) was added, followed by Hoechst 33342 (Chemometec). After a PBS wash, the cells were resuspended in Annexin V binding buffer (Invitrogen) containing 10 μg/mL propidium iodide (Chemometec). Cells were loaded into NC‐Slide A2 chambers, and the “Annexin V Assay” was run with the NucleoCounter NC-3000.

Human anaplastic astrocytoma cells (SW1783, HTB-13^™) were obtained from ATCC (Manassas, VA, USA). Neobavaisoflavone (7-hydroxy-3-(4-hydroxy-3-(3-methyl-2-buten-1-yl)phenyl)-4H-1-benzopyran-4-one), penicillin G, and dimethyl sulfoxide (DMSO) were retrieved from Sigma-Aldrich Inc. (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were acquired from Cytogen (Zgierz, Poland). Trypsin/EDTA solution was purchased from ThermoFisher Scientific (Waltham, MA, USA). Cell proliferation reagent WST-1 was obtained from Roche (Mannheim, Germany). NC-Slides™ A8 and Via1-Cassettes™, as well as Solution 3 (1 μg/mL DAPI, 0.1% Triton X-100 in PBS), Solution 7 (200 μg/mL JC-1 in DMSO), Solution 8 (1 μg/mL DAPI in PBS), Solution 15 (500 μg/mL Hoechst 33342, aqueous), Solution 16 (500 μg/mL propidium iodide, aqueous), were retrieved from ChemoMetec (Lillerød, Denmark). Neomycin sulfate was acquired from Amara (Kraków, Poland). Annexin V binding buffer and Annexin V-CF488A conjugate were obtained from Biotium (Fremont, CA, USA). In the study, the following drugs were used: doxorubicin (Doxorubicin Accord, Accord, Ahmedabad, India), etoposide (Etoposid-Ebewe, Ebewe Pharma, Ahmedabad, India), irinotecan (Irinotecan Accord, Accord, Ahmedabad, India). The rest of the chemicals were purchased from POCH S.A. (Gliwice, Poland).

Briefly, the human lung epithelial adenocarcinoma line A549 was cultured in 96-well plates (1 × 10⁵ cells/well) with S. pneumoniae bacteria or EMVs for 24 h, after which cell viability and apoptosis were measured as described previously [7 (link)]. The culture medium was RPMI 1640 (Gibco, Waltham, MA, USA) supplemented with heat-inactivated 10% FBS and antibiotics (Gibco). The ranges of multiplicity of infection (MOI) of S. pneumoniae that were used to infect the A549 cells were 0.001, 0.1, 10, and 1,000 for S. pneumoniae BAA-255 and 0.001, 0.01, 0.1, and 1 for S. pneumoniae KCCM-41569. The concentrations of S. pneumoniae BAA-255 EMVs were 50, 100, and 200 μg protein in 100 μL of culture medium. To measure cell viability, the cells were stained with acridine orange and DAPI (ChemoMetec, Allerød, Denmark). To measure apoptosis, the cells were stained with FITC-conjugated Annexin V, propidium iodide, and Hoechst (ChemoMetec) according to the manufacturer's instructions. The stained cells were then analyzed in a NucleoCounter NC-3000 image cytometer (ChemoMetec).