Microvette cb 300 lh tubes

Immediately after application of MN to shaved, hairless skin (Smooth Care^®, Hair Removal Cream, Boots Company PLC, Nottingham, UK) on the back of each rat, a theophylline solution of 2 mg/mL concentration was administered to the anaesthetised Sprague Dawley^® rats via oral gavage. Volume of the theophylline solution administered to each rat was based on their individual weight, to achieve a dose of 5 mg/kg or 10 mg/kg. Following application, MN arrays remained in situ for 1 h. Blood samples of 200 μl were collected at pre-defined time intervals by lateral tail vein puncture (Microvette^® CB 300 LH tubes, Sarstedt AG & Co. Nümbrecht, Germany). All animal experiments throughout this study were conducted according to the policy of the Federation of European Laboratory Animal Science Associations (FELASA) and The European Convention for the protection of vertebrate Animals used for Experimental and Other Scientific Purposes, with implementation of the principle of the 3Rs (replacement, reduction, refinement).

After blood was drawn by cardiac puncture, plasma was isolated using Microvette CB 300 LH tubes (Sarstedt). The concentrations of total cholesterol, LDL/VLDL cholesterol, HDL cholesterol and triglycerides were determined using enzymatic colorimetric assays (#E2HL-100 and #ETGA-200; BioAssay Systems) as per the manufacturer's instructions. Glucose levels were detected using the Contour^®Next EZ Blood Glucose Monitoring System with Contour^®Next test strips.

Blood from Fn1^syn/syn or Fn1^+/+ mice was collected in heparinized Microvette CB 300 LH tubes (Sarstedt) and platelets were counted using a ProCyte Hematology Analyzer (IDEXX Laboratories, Ludwigsburg, Germany). To isolate platelets, heparinized blood from β3^+/+ or β3^-/- mice was centrifuged at 70xg for 10 min at RT, the platelet enriched upper phase was then centrifuged at 800xg for 10 min and the platelet pellet was finally washed twice with Tyrodes buffer pH 6.5 (134 mM NaCl, 2.9 mM KCl, 12 mM NaHCO₃, 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 5 mM glucose, 0.35% bovine serum albumin (BSA)). Washed platelets were resuspended in Tyrodes buffer pH 7.4 and counted using a ProCyte Hematology Analyzer (IDEXX Laboratories). For experiments, platelet numbers were adjusted to equivalent concentrations with Tyrodes buffer pH 7.4 complemented with 1 mM CaCl₂, 1 mM MgCl₂.