Z1 particle counter

Cell number was determined for all cell types at the time of
harvest, which for MG63 cells stably silenced for integrins
α1, α2,
α5, and β1 was after
seven days in culture. At confluence, cells were released from TCPS and Ti
surfaces using two sequential incubations with 0.25% trypsin for 10 min at
37 °C to ensure that no cells remained on the rough Ti surfaces and
counted using an automated cell counter (Z1 Particle counter, Beckman
Coulter, Fullerton, CA).

All colorimetric and fluorescent assays were performed using a SpectraMax M3 Multimode Microplate Reader and analyzed with SpectraMax SoftMax Pro 6.3 software (Molecular Devices, Sunnyvale, CA). For analysis of cholesterol in cell lysates and lipid raft fractions, an Amplex Red based detection kit was used according the instructions provided by Sigma-Aldrich. Samples were compared to a standard curve generated from human low density lipoprotein prepared in either cell lysis buffer (20 mM tris-HCl, 150 mM NaCl, 5 mM EDTA, pH = 7.4) or lipid raft extraction buffer (see below). Protein content of cell lysates and lipid raft fractions was quantified using a Detergent Compatible analysis kit (BioRad, Hercules, CA) based on the Lowry method [25 (link)].
Viability was measured as a function of the ability of the cells to convert resazurin to fluorescent resorufin as previously described [26 ]. To assess growth, cell number quantified using a Beckman Coulter Z1 Particle Counter (Indianapolis, IN). At all concentrations used, pravastatin had no effect on either cell growth or viability (Supplemental Fig. 1).

Blood was obtained from 30 healthy volunteers (17 men and 13 women), who provided written consent, and were free of medications affecting platelet function for two weeks prior to venipuncture. The study protocol was approved by the Institutional Review Board of the University of Arizona (protocol# 1810013264 “Optimization Cardiovascular & Mechanical Circulatory Support (MCS) Devices Thrombogenicity for Eliminating Anticoagulation”). Fresh blood samples were anticoagulated with acid citrate dextrose solution at blood : anticoagulant ratio – 9 : 1. Platelet-rich plasma (PRP) was obtained by centrifugation of blood at 400g for 15 min at room temperature. Residual blood was spun at 1430g for 20 min at room temperature to yield platelet-poor plasma. Gel-filtered platelets (GFP) were isolated from PRP by gel-chromatography through Sepharose-2B³² , and platelet count was measured on Z1 Particle Counter (Beckman Coulter, Indianapolis, IN). Platelet-contained fractions were stored and handled at room temperature, to minimize storage-associated platelet function decline³³ . Similarly, to limit 37°C-stimulated platelet apoptosis^{34 (link),35} and associated alterations of the surface expression of platelet receptors^{12 (link)} and microparticle generation^{36 (link)}, most experiments were performed at room temperature if not otherwise specified.

Cells were seeded in 6-well plates such that triplicate measurements
could be made at each indicated time point. Media was refreshed every other
day as appropriate. Cells were trypsinized and counted using a Z1 Particle
Counter (Beckman Coulter). Phase-contrast images were captured at indicated
time points using a Zeiss Axiovert 40 CFL Microscope. Cell viability was
determined by trypan blue exclusion using a Vi-CELL XR (Beckman
Coulter).