Percolation density

Siitterlin U, Thies W-G, Haffher H, et al. 1984. Comparative studies on the lysosomal association of monomeric 239Pu and 241Am in rat and Chinese hamster liver Analysis with sucrose, metrizamide, and percoll density gradients of subcellular binding as dependent on time. Radiat Res 98 293-306. 

The molar ratio of the components of metaphase chromosome was determined by Uchiyama and his colleagues (Uchiyama et al., 2005). Metaphase chromosomes has been isolated from synchronized human cell line and then purified by sucrose density gradient centrifugation and Percoll density gradient centrifugation. The major components described in this chapter are summarized here. Molar ratio is provided per 100 histone H4 molecules. 

Petrenko, A.Yu., Grischuk, V.P., Roslyakov A.D. et al. (1992) Survival, metabolic activity and transport of potassium ions of rat hepatocytes after rapid freeze-thawing under protection of dimethylsulfoxide and separation in Percoll density gradient, Cryo-Letters. 13 87-98. 

Garson, M. J., Flowers, A. E., Webb, R. I., Charan, R. D., and McCaffrey, E. J., A sponge/dinoflagellate association in the haplosclerid sponge Haliclona sp. cellular origin of cytotoxic alkaloids by Percoll density gradient fractionation, Cell Tissue Res., 293, 365, 1998. 

To 15 mL polystyrene conical tubes add 6 mL of isotonic Percoll (density, 1,070g/mL). Underlayer with 3 mL isotonic Percoll (density, 1,079g/mL). We use a Gilson Minipuls II pump with tubing narrowed at the end which allows us to prepare 5 tubes at a time. 

Hahn, C.G. and Covault, J. (1990) Isolation of transcriptionally active nuclei from striated muscle using Percoll density gradients. Anal. Biochem. 190, 193-197. 

Figure 7. Focusing FFF fractogram of 9.87- and 40.1-iim PS latex microspheres in diluted Percoll density gradient generated by the electric potential of 32 mV and using the stop-flow procedure for relaxation. (Reproduced with permission from reference 13. Copyright 1993 Springer-Verlag.)...

If apoptosis is only occurring at relatively low levels in cultures, it may be necessary to obtain a purified population of apoptotic cells prior to DNA isolation and electrophoresis. This may be achieved by exploiting the fact that apoptotic cells are more dense than normal cells. Hence it is possible to purify apoptotic cells by isopycnic centrifugation. Percoll can be used to create solutions of different densities. The precise Percoll densities used for isolation of apoptotic cells will depend on the cell type under investigation. 

Fig. 3 Isolation of nuclei from suspension tissue culture cells. (A) Low-power SEM image of nucleus isolated from K.S62 tissue culture cell, after isolation and Percoll density-gradient purification. This particular nucleus shows surface indentations due to its position in the cell cycle (approaching prophase), but the overall surface morphology of the nuclear envelope is largely free of cytoplasmic contamination, and numerous pore complexes (arrowed) arc apparent. Scale bar, 1.0 /u,m. (B) Detail of A, showing cytoplasmic surface of the nuclear envelope with clearly visible nuclear pore complexes, and a typical density of ribosomes (remember the outer nuclear envelope is the innermost element of the rough endoplasmic reticulum). Scale bar, 125 nm.

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. 

Chloroplasts frero A. mediterranea were obtained by a very gentle isolation method combining both membrane filtration and a Percoll density gradient step. These chloroplasts exhibit unique long-term biosynthetic activities with a 002 fixation rate of 80 yumoles OO2 fixed per mg chl. x h and a photosynthetic activity of up to 65 fmoles O2 evolved per mg chl." X h (5 h after isolation). For comparison, whole Acetabularia cells show a photosynthetic capacity of 105 mmoles O2 evolved per mg chl." X h". ... 

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