Dioc6

To test chemosensitivity, 3×10⁴ NCI-H69 or NCI-H69V cells were seeded in 6 well plates for 12 h and were then treated with 200 µM Etoposide (Sigma-Aldrich, Munich, Germany) for 48 h as described before [21] (link). Afterwards, cell viability was tested using propidium iodide (PI) (Sigma-Aldrich, Munich, Germany) and 3,3′-dihexyloxacarbocyanine iodide (DIOC6) (Sigma-Aldrich, Munich, Germany). Cells were incubated in RPMI 1640 containing 0.5% BSA, 10 nM DIOC 6 and 2 mg/ml PI at 37°C for 20 min and analyzed by flow cytometry on a FACSCalibur (Becton Dickinson, Mountain View, CA, USA). Flow cytometry data were analyzed using FlowJo software v.7.6.3 (Tree Star Inc., San Carlos, CA, USA).

9 × 10⁵ cells of Ba/F3 cells stably expressing PDGFRA WT or mutant were cultured in 3 ml of medium with or without 5% serum for 16 h. Cells were collected by centrifugation and stained with 0.1 μM DIOC(6) and 100 μg ml⁻¹ PI (Molecular Probes, Life Technologies) at 37 °C for 15 min to determine mitochondrial membrane potential and late apoptosis after serum starvation for 16 h. Cells cultured in medium with 5% serum were served as control. Percentage of cells with decreased membrane potential (DIOC(6)-low) and late apoptotic cells (PI-positive) were analyzed with flow cytometry (BD FACSAriaII).

The mitochondrial transmembrane potential was measured by cytofluorimetry, using the fluorescent dye 3,3′-dihexyloxacarbocyanine iodide [DiOC6(3), Sigma Aldrich]. DiOC6(3) is a green fluorescent membrane dye that has been used to detect mitochondrial membrane potential in live cells. The fluorescence of the dye is enhanced when incorporated into mitochondrial membranes. Changes in mitochondrial membrane potential were assessed in cells by measuring fluorescence intensity due to a conformational change of the dye. The G93ADOXY− cells, G93ADOXY+, and G93ADOXY+ treated with exosomes (1 × 10⁶) were processed as described above. Cells were resuspended in PBS containing DiOC6(3) (1 nM), incubated at 37°C for 15 min, immediately analyzed by flow cytometry with a FACSCanto II (BD) and analyzed with FlowJo software (Treestar Inc.).

For monitoring the mitochondrial membrane potential, cells were washed once in PBS before addition of 60 nM DiOC₆ (3,3’-dihexyloxacarbocyanine iodide) (Sigma-Aldrich, Saint-Louis, MO, USA). Cells were then incubated for 30 min at 26°C, washed in PBS and resuspended in PBS. DiOC₆ fluorescence was measured by flow cytometry using 488 nm excitation and measuring green fluorescence emission on the BD LSRFortessa™ cell analyzer. Data were exported and analyzed with Flowjo software for evaluation of the percentage of cells with a depolarized or hyperpolarized mitochondrion and of the FSC median.

To determine variations in mitochondrial membrane potential and cytoplasmic membrane damage, HaCaT and PC3 cells were analyzed using DiOC₆ (Molecular Probes D273) and propidium iodide (PI) (Sigma P4170). The cells were suspended in 500 μL of PBS containing 5 μL of 10 μM DiOC₆ and 5 μL of 1 mg/mL of PI, and then the cell suspensions were incubated in the dark at room temperature for 20 min, washed, resuspended in PBS, and analyzed by flow cytometry (BD LSR Fortessa). The concentrations of pugnin A and B peptides were 50, 100, and 150 μM, with three repetitions [78 (link)].

Intracellular ROS levels of isolated pancreatic islet cells were measured by flow cytometry (BD Accuri Flow Cytometer) using 2,7-dichlorofluorescein diacetate (DCFDA) fluorescence dye^{63 (link)}. Following isolation, islet cells were treated with 1 μM DCFDA and incubated at 37 °C for 30 min in the dark. After staining, the cells were passed through a 40 μm cell strainer to eliminate cell clumps.
The mitochondrial membrane potential was assessed using the fluorochrome stain DiOC6 (Molecular Probes). Isolated islet cells were loaded with 40 nM DiOC6 for 30 min and then analyzed using a BD Accuri Flow Cytometer.
For cell cycle analysis, cells were fixed with chilled 70% ethanol overnight at 4 °C. Cells were washed twice with PBS at 1200 rpm for 5 min to remove ethanol and finally cells were incubated propidium iodide in dark for 30 min and then acquired on a BD Accuri Flow Cytometer with excitation at 488 nm and emission was collected by 585/40 nm band pass filter.
Apoptosis in isolated islet cells was evaluated utilizing the Annexin V fluorescent probe. This protein binds to phosphatidylserine residues exposed on the cell surface of apoptotic cells. Flow cytometry was employed to monitor apoptosis, employing Annexin V and Propidium Iodide with a 488 nm laser and detecting fluorescence in FL1 (530/30) and FL2 (585/40) channels.