Ml open top polyallomer tube

A discontinuous iodixanol gradient was used as described by (12 (link)) with some modifications. Solutions of 5, 10, 20 and 40% iodixanol were made by mixing appropriate amounts of a homogenization buffer [0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCL, (pH 7.4)] and an iodixanol working solution. This working solution was prepared by combining a working solution buffer [0.25 M sucrose, 6 mM EDTA, 60 mM Tris-HCl, (pH 7.4)] and a stock solution of OptiPrep™ (60% (w/v) aqueous iodixanol solution). The gradient was formed by layering 4 mL of 40%, 4 mL of 20%, 4 mL of 10% and 3.5 mL of 5% solutions on top of each other in a 16.8 mL open top polyallomer tube (Beckman Coulter). One millilitre CCM sample was overlaid onto the top of the gradient which was then centrifuged for 18 hours at 100,000 g and 4°C (SW 32.1 Ti rotor, Beckman Coulter). Gradient fractions of 1 mL were collected from the top of the gradient, diluted to 16 mL in PBS and centrifuged for 3 hours at 100,000 g and 4°C. The resulting pellets were resuspended in 100 µL PBS and stored at −80°C. To estimate the density of each fraction, a standard curve was made of the absorbance values at 340 nm of 1:1 aqueous dilutions of 5, 10, 20 and 40% iodixanol solutions. This standard curve was used to determine the density of fractions collected from a control gradient overlaid with 1 mL of PBS.