Dioc6

Microfluidic flow assay was performed as previously described (Halaidych et al., 2018 (link)). Briefly, Vena8 Endothelia+ chips (Cellix) were coated with 50 μg/mL fibronectin overnight at 4°C. ECs were first treated with 10 ng/mL BMP9 (R&D) for 24 h, then stimulated with TNF-α (10 ng/mL) for 12 h (overnight) in the presence of BMP9. Next day, ECs were collected and injected into the microfluidic channel. Then, the chip was incubated at 37°C to facilitate cell attachment. Monocytes were collected and stained with DiOC6 (1:5,000) (Sigma), then resuspended in IF9S medium at the end concentration of 2.5 × 10⁶ cells/mL. For flow experiments, monocytes were perfused for 5 min at 0.5 dyn/cm² through the microfluidic channel, followed by a 5 min wash with IF9S medium. The number of adherent fluorescently labeled monocytes on ECs was quantified using the open source software CellProfiler (Carpenter et al., 2006 (link)).

Chemokines were diluted in calcium-free PBS, pH 7.4, at their final concentrations (MIF: 16 nM; CXCL4L1: 32 nM) and allocated into separate reaction tubes. 200 µL of each solution were distributed onto separate collagen-coated cover slips (100 µg/mL) and incubated for 2 h. Cover slips were blocked with PBS, pH 7.4, containing 1% BSA for 1 h. Next, human whole-blood was diluted at a 5:1 ratio with PBS, pH 7.4, containing calcium. Before perfusion, the blood was incubated with fluorochrome 3,3′-dihexyloxacarbocyanine iodide (DiOC₆, 1 mM; Sigma Aldrich) for 10 min at RT. Thereafter, the blood was allocated into 1 mL syringes and perfused over the different cover slips, through a transparent flow chamber with high shear rate (1000 s⁻¹) for 5 min. Per run, one 2-min video clip was recorded (200 ms/frame, Nikon Eclipse Ti2-A, 20 × objective). Afterwards, the chamber was rinsed and pictures were taken of five representative areas using the same objective. The covered area was analyzed using the NIS-Elements AR software (Nikon) and the mean percentage of the covered area, the mean thrombus area as well as the mean thrombus count were determined.

The mitochondrial membrane potential was measured with DiOC6 (3, 3′-dihexyloxacarbocyanine iodide; Sigma), a fluorochrome that is incorporated into the cells depending upon the Δψ_m. Loss of DiOC6 fluorescence indicates reduction in the mitochondrial inner transmembrane potential, which was monitored using flow cytometer as described before. In brief, FQ treated MIA PaCa-2 and Panc-1 cells were stained with DiOC6 at a final concentration of 40 nM for 30 min at 37 °C in dark. Cells were washed, and the fluorescence intensity was analysed by a flow cytometer (Guava Technologies). A minimum of 5000 events were counted.

For a given dose, the number of alpha-particle tracks traversing the cell or nucleus is dependent on its area, and at low doses not all cells are necessarily traversed [32 (link)]. Thus, measurements of the cellular and nuclear area were made to calculate the average number of alpha-particle traversals per cell and per nucleus, by different doses.
Immediately prior to irradiation, two randomly chosen spare Hostaphan-based dishes of HF19 cells were stained with DIOC6 (3, 3′-dihexyloxacarbocyanine iodide, Sigma), a fluorescent dye which stains the cell mitochondria, endoplasmic reticulum and vesicle membranes. Random ‘saved’ multiple cell images were taken of horizontal sections across these stained monolayer cells. The images were taken by a confocal laser scanning microscope (BioRad-Lasersharp 2000 confocal microscope coupled to a Nikon TE2000 microscope with an ion argon laser operating at 488 nm wavelength). This allowed subsequent measurement of the living cells’ nuclear area and cellular area as shown in Figure 1. The computer programme ‘Image J’ was initially used to manually draw around the circumference of each nucleus and cell, by which the computer is able to calculate the area of each, as shown in Figure 1.