Visiview

All values are given as mean ± SEM or SD. Data were analyzed using VisiView (Visitron), Microsoft Excel (Microsoft), Igor Pro (Wavemetrics), Image Lab™ (Bio-Rad) and GraphPad (GraphPad Software Inc.). Rate constants (k values) of iso-cells were calculated for each trace by exponential decay analysis after removal of external Ca²⁺. Significances of data were calculated with an unpaired two-sided Student’s t-test if Gaussian distribution was given. If no Gaussian distribution was given, data were analyzed with the nonparametric Mann-Whitney test. For multi-parameter analysis data were analyzed with ANOVA. Degrees of significance were set at * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

In offline video analysis video analysis, the software VisiView (Version 3, Visitron Systems GmbH, Puchheim, Germany) was used. Platelets and leukocytes were quantified by respective characteristics and in accordance to rheological properties classified as (a) free flowing (no intercellular or endothelial contact), (b) rolling (slower than centerline blood flow) and (c) adherent (sticking to endothelium for more than 30 s) [46 (link)]. Rolling velocity was expressed as number of rolling cells per second per millimeter of vessel diameter (cells/s/mm), whereas adhesion was presented as number of sticking cells per square millimeter of vessel surface calculated by diameter and length (cells/mm²). Interactions between platelets and leukocytes, platelets and endothelial cells, leukocytes and endothelial cells as well as all three types of cells were documented independently.

The intracellular pH of PANC-1 cells was measured using fluorescent live-cell imaging (Axiovert TV100, Zeiss, Oberkochen, Germany) as described previously [45 (link)]. Cells were loaded with the fluorescent pH indicator 2′7′-bis(carboxyethyl)-5-carboxyfluorescein (BCECF-AM) (3 µM) for up to 2 min. The excitation wavelength alternated between 490 nm and 440 nm. The emitted fluorescence was detected at 510 nm. The mean fluorescence of each cell was measured in 10 s intervals. Data acquisition and the polychromator (Visitron Systems, Puchheim, Germany) were controlled by the program VisiView (Visitron Systems). The cells were superfused with prewarmed (37 °C) CO₂/HCO₃⁻-buffered Ringer’s solution (116 mM NaCl; 24 mM NaHCO₃; 5.4 mM KCl; 0.8 mM MgCl₂; 1.2 mM CaCl₂; 5.5 mM Glucose) at pH 7.4. NaHCO₃ was lowered to 4.7 mM for pH 6.6. pH measurements were calibrated with a two-point calibration (130 mM KCl; 1.2 mM CaCl₂; 0.8 mM MgCl₂; 10 mM Hepes; 5.5 mM Glucose; pH 7.5 and pH 6.5; supplemented with 10 µM Nigericin) (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). For data analysis, the mean fluorescence intensity of the cell area was measured and corrected for background fluorescence. Afterward, the 490 nm/440 nm ratio was determined, and the pH_i was calculated with a linear regression of pH 6.5 and 7.5.

Cells were placed onto 2% (w v⁻¹) agar cushions and observed using an IX83 motorized inverted microscope (Olympus, Hamburg, Germany), equipped with a PlanApo × 100/1.45 oil TIRF objective (Olympus) and a VS-LMS4 Laser-Merge-System with solid-state lasers (488 nm/ 70 mW and 561 nm/ 70 mW, Visitron System, Puchheim, Germany). For photo-bleaching experiments, a 405 nm/ 60 mW diode laser was used, which was coupled into the light path by an OSI-IX 71 adaptor (Visitron System) and controlled by a UGA-40 controller (Rapp OptoElectronic, Hamburg, Germany) and VisiFRAP 2D FRAP control software. Z stacks were generated by using an objective piezo (Piezosystem Jena GmbH, Jena, Germany). Images were acquired using a Photometrics CoolSNAP HQ2 camera (Photometrics/ Roper Scientific, Tucson, USA). All parts of the system were under the control of the software package VisiView (Visitron System). Samples were observed for no longer than 10 min, to prevent oxygen depletion. All image processing was performed in MetaMorph 7.8.x (Molecular Devices, Wokingham, UK).