μ slide vi0.4 chamber

Clots were formed from 30 % FDP, 16 µM phospholipids (Rossix, Molndal, Sweden) and 0.25 µM AlexaFluor 488 labelled fibrinogen (ThermoFisher Scientific, Waltham, USA) in 10 mM Tris pH 7.4, 140 mM NaCl, 0.01% TWEEN-20. FDP was substituted with PNP for a control. Cryoprecipitate and Fg-C were added at a range of concentrations—0.5, 2 and 3 mg/mL. Clotting was initiated by the addition of 0.1 U/mL thrombin (Sigma Aldrich, St Louis, MS, USA) and 10.6 mM CaCl₂. Clots were polymerised in an Ibidi μ-slide VI^0.4 chamber and incubated for 2 h at 37 °C in a moist box. Clots were imaged using a ×63 1.4 oil immersion objective and Zeiss 710 laser scanning confocal microscope. Images were recorded on differential interference contrast (DIC) microscopy and at excitation wavelengths of 488 nm and analysed using Zen 2012 SP1 v8.1 (Black edition).

Oscillatory fluid shear stress (OFSS, τ = ± 10 dyne/cm²) at 1 Hz was applied over a cell monolayer. The fluid flow setup consisted of μ–slide VI^0.4 chamber (ibidi GmbH, Germany), Legato 200 syringe pump (KD Scientific, MA, USA) and a syringe filled with flow media (phenol free α-MEM supplemented with 1% FBS and 10mM Hepes) that is optimized for real-time imaging. Culture media in μ–slide VI^0.4 chambers was first replaced with flow media followed by flow-loop set up and equilibrated under static condition for 2 min prior to OFSS. Aliquot (50 μl) of static media was collected right after 2 min equilibration time and flow conditioned media was collected at the end of 5 min OFSS exposure for measurements of cellular ATP release.
To inhibit P2X7R and Panx1 function, cells were pretreated with the P2X7R blockers A438079 hydrochloride (10 μM; TOCRIS bioscience, Bristol, United Kingdom) [41 (link), 51 (link)], A740003 (10 μM; TOCRIS) [30 (link), 52 (link)] and mefloquine (MFQ, 100nM; QU024-1, Bioblocks, San Diego, CA) [26 (link)] in flow media for 15 min prior to OFSS and during 5 min of OFSS.