Eclipse ti2 a

Cells were incubated with 50 μM EdU solution for 3 h and then fixed using 4% paraformaldehyde. The Edu was stained with Apollo 567 and Hoechst 33342 was applied to stain the cell nuclei. Cells were detected using Eclipse Ti2-A fluorescence microscope (Nikon, Japan).

Isolated murine platelets in Tyrodes buffer (pH 7.4) were supplemented with 1 mM CaCl₂, activated with 1 µg/ml CRP-XL (CambCol, Cambridge, UK), and incubated on fibrinogen-coated (100 µg/ml; Sigma Aldrich Co., St. Louis, MO, USA) coverslips for 30 min at room temperature. Afterward, platelets were fixed for 15 min with 4% paraformaldehyde (Sigma Aldrich Co., St. Louis, MO, USA) and washed three times with PBS (Sigma Aldrich Co., St. Louis, MO, USA). The coverslips were mounted onto slides and several images from randomly selected areas were taken (Nikon Eclipse Ti2-A, 100x DIC objective). The images were analyzed with the NIS-Elements AR software (Nikon, Japan). The classification of platelets into spreading stages was performed manually. Platelets on five randomly selected images per condition were counted.

Light microscopy images were taken on Nikon Eclipse Ti2-A in phase-contrast configuration, equipped with a Hamamatsu ImageEM-X2 CCD camera and a 20 x objective. For timelapse images, we used the microscope control software µManager (Stuurman et al., 2007 (link)) and acquired an image every 5 min. The phase-contrast images were analysed with the software Fiji (Schindelin et al., 2012 (link)). Fiji was also used to produce the three-dimensional renderings of the biofilm from the confocal images using the temporal color code function. For the fluorescent visualizations, we used a Nikon Eclipes T1 inverted microscope coupled with a Yokogawa CSU-W1-T2 confocal scanner unit and equipped with an Andor iXon Ultra EMCCD camera. The images were acquired with a 60 x water immersion objective with N.A. of 1.20. We used Imaris (Bitplane) for analysing and producing cross-sections of the z-stacks.

Histopathological examination was performed according to the reference with minor modifications [32 (link)]. Briefly, liver tissues were fixed in 4% PFA for 24 h, dehydrated by gradient ethanol, paraffin-embedded, sectioned (~4 μm), stained with H&E, and mounted with neutral gum. The morphological changes in tissues were observed under an optical microscope (Nikon Corporation, ECLIPSE Ti2-A, Tokyo, Japan) and photos were taken (magnification, 200×).

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.

Human whole-blood was incubated with the fluorochrome 3,3′-dihexyloxacarbocyanine iodide (1 mM DiOC₆, Sigma Aldrich Co., St. Louis, MO, USA) for 10 min at room temperature. Then 1 ml of the blood was perfused over a collagen-coated surface (100 µg/ml), through a transparent flow chamber with high (1000 s⁻¹) shear rates. In contrast, murine blood was anti-coagulated with heparin and diluted in Tyrode´s buffer, supplemented with 1 mM CaCl₂, and was either stained with DiOC₆ for 10 min at room temperature or used directly. Afterward, the blood was perfused through a transparent flow chamber over a collagen-coated surface (shear rate = 1000 s⁻¹). During the perfusion 1 min videos were taken (1 s/frame, Nikon Eclipse Ti2-A, ×20 objective) for the murine as well as human experimental set-ups. Afterward, the chamber was rinsed, and pictures were taken of five representative areas (Nikon Eclipse Ti2-A, 20x objective). The covered area was analyzed using the NIS-Elements AR software (Nikon, Japan), and the mean percentage of the covered area was determined. In the case of agonist treatment, the blood was divided into samples, which were subsequently incubated for 30 min at room temperature either with an ACKR3 agonist or a vehicle control at the indicated concentrations.