Percoll gradients preparation

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

Membrane separation. Cells were harvested and washed in a medium containing 10 mM Tricine/NaOH, 10 mM NaCl, 300 mM sucrose and once passed through a French-pressure cell at 8.1 MPa. Intact cells and large cell debris were collected at 5000 g for 10 min the pellet obtained at 20000 g was brought on top of a discontinuous Percoll gradient, prepared in the isolation medium and after centrifugation at 5000 g for 30 min three bands appeared at the 15, 20 and 30% border lines. The bands at 15 and 20% contained the thylakoid membranes and the band at 30% a composite of cell wall and cell membrane. These preparations were used for further identification. 

We routinely use a (dedicated) laminar flow cabinet to prepare the Percoll gradients and perform subsequent manipulations. It is important to avoid activating the cells during these procedures. 

We routinely obtain about 500 x 10 cells from a rat weighing 200 g. The viability is about 90%. In the absence of DNAse in Step 8, the viability is nearer 80%. To obudn a hepatocyte population with a viability above 95%, the hepatocyte preparation can be centrifuged in a Percoll gradient (4). 

As shown in Table 1, crude mitochondrial fractions prepared by differential centrifugation (as other slightly different conventional methods) appeared to exhibit pronounced aryl-ester hydrolase (EC 3.1.1.2) activity. As this activity was found specifically in microsomes, mitochondrial fractions prepared conventionally are always contaminated by microsomes. In mitochondrial fractions isolated on Percoll gradient imder the conditions precisely described in the Materials and Methods section, the above microsomal activity was extremely low. The pattern was similar for acyl-CoA Hgase (ACL), its specific activity was very high in microsomal fractions and almost negligible in PercoU-purified mitochondrial fractions (Fig. 1). Taking into account the specific activity of aryl-ester hydrolase in microsomal fractions, the amount of microsomal protein present in purified mitochondrial fractions could be estimated around 2% and was sufficient to account for the residual ACL activity in these fractions. It was, therefore, clear that a totally pure liver mitochondrial fraction could not esterify fatty acids with CoA. 

Add the cell homogenate gently on top of the prepared Percoll gradient. 

Preparation of envelope membranes. Envelope membranes were prepared from purified intact chloroplasts isolated from 2 to 3 week old pea seedlings using methods described by Cline et al. (1981). In brief, purified intact chloroplasts were prepared by Percoll gradient centrifugation and repeated differential centrifugation. Envelope membranes were prepared from hypotonically shrunken, freeze/thaw ruptured chloroplasts by flotation to the 0.3 M/1,2 M sucrose interface on a step sucrose gradient,... 

For preparation of a density gradient with a large linear range, manufacturers give the following conditions Spin Percoll in 0.15 M NaCl (final concentration) with at least 10 000 x gj,, for Percoll in 0.25 M sucrose apply 25 000 x g. Use near vertical or vertical rotors for cell separation. 

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. 

Mouledous, L., Hunt, S., Harcourt, R., Harry, J.L., Williams, K.L. and Gutstein, H.B. (2003) Proteomic analysis of immunostained, laser-capture microdissected brain samples. Electrophoresis 24,296-302. Nagy, A. and Delgado-Escueta, A.V. (1984) Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material (Percoll). J. Neurochem. 43, 1114-1123. 

PRP and PPP preparation. If rats or mice are used, they are anaesthetized by an i.p. injection of sodium pentobarbital (50 mg/Kg), and blood is drawn into heparin (5 units/ml final) or citrate/dextrose (final concentration 0.38% and 0.48% (wt/vol), respectively) anticoagulant from the dorsal aorta or by heart puncture, with a 22-gauge needle (the choice of heparin as anticoagulant appears to be more appropriate, because chelation of divalent cations by trisodium citrate or EDTA might affect platelet-tumor cell interaction). Blood is then diluted with an equal volume of 0.9% NaCl, and centrifuged for 7 min at 400 x g on preformed gradients of 70% Percoll containing 0.9% NaCl. The yellowish supernatant is the PPP, while PRP forms a white band between PPP and the percoll layer. Platelets in PRP are counted with a Coulter counter and diluted with PPP to a concentration of 10 /ml. PPP is a source of prothrombin, and should thus be included in the assay. 

A column of CM-Sephadex (2.5x20 cm, Pharmacia Biotech, Uppsala, Sweden) is prepared according to the manufacturer s instruction by using 10 mM PB, pH 6.0, as an equilibration buffer. Apply the dialyzed fraction to the column, wash with 200 ml of 10 mM PB, pH 6.0 (this percolate contains component I), and elute component II with a gradient from 0 to 0.4 M sodium chloride in 1000 ml of 10 mM PB, pH 6.0. Collect the fractions containing component II and concentrate by ultrafiltration through a P0200 membrane (UHP-43K, Advantec Co., Tokyo, Japan). 

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... 

Nagy, A., and Delgado-Escueta, A. V., (1984). Rapid Preparation of Synaptosomes from Mammalian Brain Using Nontoxic Isoosmotic Gradient Material (Percoll), ,/ Neurochem. 43 1114-1123. 

Nagy, A. and Delgado-Escueta, A V (1984) Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material (Percoll) J Neurochem. 43, 1114—1123. 

Subcellular fractions were prepared by standard differential and density gradient centrifugation protocols. Purified fractions of mitochondria and peroxisomes were prepared by Percoll and Nycodenz centrifugation, respectively. The following marker... 

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. 

Although these hPT cells are predominantly (>85%) of PT origin, a small degree of contamination with other epithelial cell populations is unavoidable and, therefore, will exist. While our laboratory has typically not performed additional purification procedures with the hPT cells, options are available to further enrich the PT cell preparation. For example, density gradient centrifugation of the renal cortical cells on Percoll can be performed to obtain PT cells that are >95% of PT origin and distal tubular (DT) cells that have minimal contamination with cells of the PT region, as we have done with cortical cells from rat kidneys (Lash and Tokarz, 1989 Lash et al., 1995). 

Percoll Prepare 80 and 30 % percoll suspension by mixing 100 % percoll with 5x gradient buffer. Use ddH20 to make up to desired volume. 

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