Percoll gradients

Fig. 2 SDS-polyacrylamide gel electrophoresis of T. vaginalis hydrogenosomes purified by isopycnic centrifugation on a Percoll gradient. 1 PFOR 2 malic enzyme, 64-kDa hy-drogenase, and Cpn 60 3 succinyl CoA synthetase fi subunit 4 Hmp35 5 succinyl CoA synthetase a subunit 6 Hmp31 (ATP/ADP carrier) 7 adenylate kinase 8 thiol peroxidase. Well-resolved but unmarked bands mostly belong to malic enzyme fragments or unknown proteins. Proteins were identified by mass spectroscopy. The 12% gel is stained with Coomassie Brilliant Blue R 250 (authors original)...

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

Carefully layer 5 mL of peripheral blood anticoagulated with 0.01 M EDTA above the Percoll gradient. 

This method of purification yields 20-50% of the total peripheral blood basophils with a purity of 60-90%. Other methods of basophil purification have previously been described. One such method involves Percoll gradient centrifugation followed by selection of IgE-i- cells by passing them through an allergen (penicil-lin)-containing column (10). This method yields 28-64% of the total peripheral blood basophils of 92% purity. 

Due to the low number of eosinophils in peripheral blood, especially in guinea pigs, eosinophils are elicited in the peritoneal cavity by repeated injection of horse serum. Eosinophils are then purified to >95% by use of a discontinous Percoll gradient. The yield of eosinophils varies but we usually use 1 exbreeder donor for every 2 4 recipients. 

Gently pour off the supernatant from the centrifuged cells and resuspend the pellet in 3 mL HBSS/BSA. Using a sterile plastic pastette, carefully layer the cell suspension onto the Percoll gradient by adding to the side wall of the tube. 

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. 

Physcomitrella patens (moss) Cultures gametophyte tissue Percoll gradients Methods for nuclear DNA, cpDNA, and mtDNA 27... 

Nicotiana tabacum, Cartha-mus tinctorius L., Nicotiana - Salpiglossus Differential centrifugation, self-generated Percoll gradients uses leaves Yields cpDNA and mtDNA 31... 

Percoll gradient-centrifuged capacitated mouse sperm have in- 118. creased fertilizing ability and higher contents of sulfogalactosyl-glycerolipid and docosahexaenoic acid-containing phosphatidylcholine compared to washed capacitated mouse sperm. Biol. 119. Reprod. 2005 72 574-583. 

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). 

Elute the different bands from the Percoll gradients. Morphological examination of these bands indicates which band is enriched for apoptotic cells. This provides an estimate for the density of the apoptotic cells. 

Adjust the Percoll gradients, placing the solution with the estimated density of apoptotic cells at the bottom of the tube. 

Figure 4.4.4 shows a typical Percoll gradient for isolation of apoptotic cells from a human promyelocytic leukaemic cell culture (HL-60). 

Placental venous endothelial cells have different characteristics depending on which part of the vasculature they have been isolated from, Tsolation, purification, and culture of primary human placental endothelial cells (HPECs) from microvessels on the venous side were reported by Jinga and coworkers (2(KX)). To gain pure cultures of HPECs, trypsin perfusion of the placental cotyledon was followed by Percoll gradient and sequential trypsinization of cultures, The,se cultures represent pure HPEC form microvessels by expressing typical patterns of markers ACE, von Willcbrand factor (typi-... 

T. gondii. Neutrophils were purified from the bone marrow of C57BL/6 mice using a Percoll gradient, then cultured with hve tachyzoites (TZ 1 2 ratio of parasites to cells) and soluble tachyzoite lysate antigen (STAg 25p,g/ml). After 18h of culmre, supernatants were collected for cytokine ELISA. Med = Medium. 

Figure 17 Facing page) Comparison of the subceUular distribution of HPMA copolymer-galactosamine (58) and cationic HPMA copolymers containing pendant side-chains terminating in oxyethy-trimethylammonium chloride (56), structure in panel (a). The profiles shown in panels (b) and (c) show rat liver fractionation using a percoll gradient at various times after iv administration of the conjugates. Panel (b) shows the distribution of I-labeled cationic HPMA copolymers at -e-10 min, -d- 20 min and -c- 60 min. Aiyl sul-fatase distribution (lysosomes) is shown in -f-. Panel (c) shows the distribution of I-labeled HPMA copolymer-galactosamine in this case, -g-10 min, -f- 20 min and -c- 60 min. Arylsulfatase distribution (lysosomes) is shown in -a- and 5 -nucleotidase (plasma membrane) in -C-.

Record, M., et al. (1985). A Rapid Isolation Procedure of Plasma Membranes from Human Neutrophils Using Self-generating Percoll Gradients Importance of pH in Avoiding Contaminations by Intracellular Membranes, BBA 819 1-9. 

The quantities given are sufficient to produce three Percoll gradients (see Note 2). 

Using a Pasteur pipet, carefully layer 4 mL of this mixture onto each of three 10/23% Percoll gradients (see Notes 2 and 5). 

Three distinct bands of material will separate on the Percoll gradients. At the top, a band comprised primanly of myelin will form. A second band, comprised of undefined cell fragments and empty sacs (the B-band), will form at the 7 5/10% Percoll boundary. The C-band, appearing at the 10/23% Percoll boundary, contains the synaptosomes. Mitochondria and large synaptosomes comprise the pellet (designated P see Figs. 1 and 2). 

Dunkley, P. R., Heath, J. W, Harrison, S. M., Jarvie, P E., Glenfreld, P J., and Rostas, J. A P. (1988) A rapid Percoll gradient procedure for isolation of synaptosomes directly from an SI fraction homogeneity and morphology of subcellular fractions. Brain Res. 441,59—71. 

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