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 VI0.4 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 VI0.4 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.
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.
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