Flow chamber

Flow chambers (μ-slide, Ibidi) were coated with rhVCAM-1/Fc Chimera (R&D Systems, 862-VC) + recombinant CXCL12 (R&D Systems, 350-NS) (1 μg/ml and 100 ng/ml in PBS, respectively) for 3 hours at RT, rinsed, and equilibrated with RPMI 1640 for 10 min at 37°C and then loaded with T cells for 10 min at 37°C. A flow rate of PBS (0 to 50 ml/min at 37°C) was applied through the temperature-controlled chamber for 92 s using a computer-driven syringe pump (SP210iW, World Precision Instruments) synchronized with image acquisition (three images per second) using an inverted transmission microscope (Axio Observer D1; Zeiss, 10×/0.3-NA objective) and Micro-Manager software (48 ). Images were analyzed using Fiji software (49 (link)) and the Cell Counter plugin. Forces necessary for cells to detach were calculated by determining the flow rate at rupture as described (16 (link)).

GCaMP 3 (gift from Michael Laflamme Lab, Toronto)^{44 (link),47 (link)} and H9, CF01 iPSC were differentiated to cholangiocyte cysts and plated down in Lab-Tek Chamber Slide (Millipore) or in flow chamber (ibidi) 2 days prior to the assay. Day 27 hepatoblasts were aggregated and plated down 2 days prior to the assay. Chambers were pre-coated with fibronectin. The cells were replaced in Tyrode buffer before the assay. H9-derived-cholangiocytes and CF01 derived cholangiocytes were stained with 10 μM Fluo4 (Invitrogen) with 0.04% Pluronic F127 (Invitrogen) for 30 min prior to the assay. To measure calcium response to ATP and TUDCA (Sigma) in 3D cysts, GCaMP derived cholangiocyte 3D cysts were embedded in the type1 collagen gel just before the assay.
Calcium response to 20 μM ATP (Sigma) or flow (Perista BioMini Pump: ATTO) were analyzed using a confocal fluorescence microscope (NIKON A1 Resonant Confocal Microscope) and images captured using the Nikon Elements software.

Twitching motility assays of N.meningitidis were performed inside a flow chamber (Ibidi GmbH, München, Germany). Bacteria (2.5x10⁷) were introduced into the flow chamber and incubated for 30 min at 37°C. Unbound bacteria were removed by 3 washes. Bacterial motility was monitored by video microscopy over a 2-minute period with the acquisition of 2 frames per second. Cell tracking was then analyzed with the ImageJ software using the spot tracking plug-in (http://icy.bioimageanalysis.org). Velocities of single bacterial tracks were calculated in time intervals of 2 s.

Blood from 12- to 20-week-old cGi500-L1 mice was perfused through a flow chamber (ibidi) for thrombus formation on a collagen-coated surface as described above. Platelet thrombi were loaded with 2.5 µM Fura-2/AM in platelet Tyrode buffer for 45 min at room temperature. A combination of filter and mirror sets was used to allow simultaneous recording of Fura-2/Ca²⁺ and FRET/cGMP signals at room temperature. An Oligochrome light source (TILL Photonics) was used to alternately excite Fura-2 at 340/26 nm and 387/11 nm, and the CFP of cGi500 at 445/20 nm. CFP and YFP emission were recorded with a CCD camera (Retiga2000R, Photometrics). Fluorescence channels were separated using a 470 nm dichroic mirror together with a beam splitter (DualView DV^{2 (link)}; Photometrics) equipped with a 516 nm dichroic mirror and CFP and YFP emission filters (480/30 nm and 535/40 nm, respectively). In each acquisition cycle, cells were sequentially excited at 340/26 nm, 387/11 nm and 445/20 nm and emitted light was recorded at 480/30 nm and 535/40 nm. Images were acquired at 0.2 or 1 Hz. For Ca²⁺ imaging of Fura-2 loaded mouse or human platelets not expressing the cGMP sensor, the same setup was used, but without the beam splitter. Samples were sequentially excited at 340/26 nm and 387/11 nm, and Fura-2 emission was recorded using a 410 nm dichroic mirror and 440LP filter^{41 (link)}.