Percoll gradients solution preparation

Percoll Gradient Preparation Percoll Stock Mix 180 mL Percoll (Pharmacia, Piscataway, NJ), 18 mL 10X Hanks Balanced Salt Solution (HBSS) without Ca,... 

Mitochondria were further enriched on a gradient of Percoll by centrifugation in polyallomer tubes in a Beckman SW28.1 rotor. Percoll-containing solutions were prepared by mixing Percoll (100%) with Buffer D (1.28 M... 

The difference in the density of the two Percoll solutions is 1% so small differences will affect the results. We have also noted that leaving the blood at room temperature for any extended time before adding it to the gradient causes basophils to localize in the lymphocyte/monocyte layer. To prevent this problem, we collect the blood into EDTA (final concentration 0.01 M) after we have prepared the required number of tubes containing the two Percoll layers. 

The previous protocol can be interrupted at an intermediate stage after the first mitochondrial pellet (step 1 crude mitochondria pellet) or after the second one (step 3 pine mitochondria pellet). In both cases, mtDNA is extracted by SDS lysis of mitochiondria followed by phenol extraction (see below for a simplified preparation of phenol see also in section HI A the minipreparation of mtDNA with a similar principle). If mtDNA is contaminated, the optional step described above can be added or an alternative solution can be chosen DNase or digitonin treatment can be tried. These various protocols are described below. A protocol similar to the sucrose gradient but-involving Percoll density gradient, often of better efficiency, is described first in the next section. 

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