Fractionation of particles is often improved by bottom-loading of a sample and this technique was used by Zhou et al [1] who described the flotation of liver mitochondria through a discontinuous iodixanol gradient of 1.175 and 1.079 g/ml, from a dense solution (approx 1.2 g/ml). 

The method provides very good resolution of the mitochondria from both lighter and denser organelles. Choi et al [2] used a similar flotation strategy for HEK293 mitochondria but based the method on one for the purification of yeast mitochondria by Meeusen et al [3]. 

Top-loading may however be preferred because of the known sensitivity of mitochondria to hydrostatic pressure [4]. Graham et al [5] first reported the use of a compromise median-loaded Nycodenz® gradient in which the LMF in 25% Nycodenz®, was underlayered by 40% Nycodenz® and overlayered by 23%, 20%, 15% and 10% Nycodenz®. The gentle centrifugation conditions of 52,000 g for 1.5 h might also be well suited to the isolation of organelles. 

While there was good separation of mitochondria from lysosomes and lysosomes from Golgi membranes, resolution of mitochondria from peroxisomes was relatively poor. This was remedied by the introduction of additional layers of 30% and 34% Nycodenz® [6], which allowed the peroxisomes to band at the 34%/40% Nycodenz® interface, later confirmed by Teoh et al [7].

Material at the 25%/34% interface was rich in cytochrome oxidase but completely lacking in peroxisome and ER markers. The strategy is satisfactory for both tissues and cultured cells and it has been adapted to the use of iodixanol [8]. For convenience the methodology below is based on the use of Optiprep™.