Calcium phosphate method

The calcium phosphate method was first used in 1973 to introduce adenovirus DNA into mammalian cells [3]. DNA-Calcium-phosphate complexes are formed by mixing DNA in a phosphate buffer with calcium chloride. These complexes adhere to the cell membrane and enter the cytoplasm by endocytosis. Disadvantages of DEAE-dextran and calcium phosphate transfection are a certain level of cytotoxicity, a complicated transfection procedure, and the fact that not all cell types can be transfected using these methods. 

The best known transfection technique using chemicals is the calcium phosphate method. In this method, calcium chloride and sodium phosphate are mixed together with DNA. Calcium phosphate crystals are formed upon combination of the chemicals and these crystals bind to and precipitate the DNA onto the cells in a... 

In order to generate the Ad5 FGF-4 virus, plasmids pACSR/FGF-4 and pJM17 were then co-transfected into HEK 293 cells using a calcium phosphate method. The... 

Dictyostelium discoideum cells can be transformed with plasmid DNA by convenient methods. The first attempts at this, using the calcium phosphate method, were reported in the early 1980s [17, 18]. Protocols were subsequently optimized by Firtel and coworkers [19, 20] and modified in many laboratories (e.g. Ref [21]). Up to 2000 transformants can be recovered from 10 D. discoideum cells [20], with electroporation protocols having been reported [22, 23]. It has also been noted that the choice of transformation method can strongly influence the yield of heterolo-gously expressed proteins [24]. Hence, both calcium co-precipitation and electroporation should be compared to generate optimized expression strains for the production of biopharmaceutical proteins in D. discoideum. 

B35. Bowness, J. M., Fractionation of acidic mucopolysaccharides on columns containing celite and calcium phosphate. Methods Carhohyd. Chem. 5, 18-20 (1965). 

HEK293T cells are transiently transfected by calcium phosphate method with plasmids encoding the G protein biosensors and CCR5-pcDNA3. 

HEK293T cells, grown until 60-80% confluence in 10-cm cell culture dishes, are transiently transfected using a modified calcium phosphate method (similar to the commercial MBS kit from Stratagene, La Jolla, CA). For transfection of each dish, a total of 15 //g of plasmid DNA (4 /ig... 

M. Jordan and R Wurm, Transfection of adherent and suspended cells by calcium phosphate. Methods, 33,136—143 (2004). 

White phosphorus may be made by several methods. By one process, tri-calcium phosphate, the essential ingredient of phosphate rock, is heated in the presence of carbon and silica in an electric furnace or fuel-fired furnace. Elementary phosphorus is liberated as vapor and may be collected under phosphoric acid, an important compound in making super-phosphate fertilizers. 

One method of treatment is to inject calcitonin, which decreases blood Ca " concentration and increases bone calcification (33). Another is to increase the release of calcitonin into the blood by increasing the blood level of Ca " ( 4). This latter treatment is accompHshed by increasing Ca " absorption from the intestine requiring dietary calcium supplements and avoidance of high phosphate diets. The latter decrease Ca " absorption by precipitation of the insoluble calcium phosphate. 

Results were obtained on the calcium phosphate growth on phosphory-lated chitin fibres using the urea/H3P04 method and subsequently soaked in saturated Ca(OH)2 solution and in simulated body fluid solution. 

The in situ precipitation route towards obtaining composites of polymer and calcium phosphate is similar to the strategy employed in naturally occurring biocomposites and well may prove a viable method for the synthesis of bone substitutes. 

It is the formation of this material which makes the reaction have a low atom economy and, owing to the cost of disposal (usually by conversion to calcium phosphate and disposal as hazardous waste), has limited its commercial usefulness to high value products. Several methods have been developed to recycle (Ph)3PO into (Ph)3P but these have proved more complex than might be expected. Typically the oxide is converted to the chloride which is reduced by heating with aluminium. Overall this recovery is expensive and also produces significant amounts of waste. 

Classical gene transfer methods still in use today are diethylamino ethyl (DEAE)-dextran and calcium phosphate precipitation, electroporation, and microinjection. Introduced in 1965, DEAE-dextran transfection is one of the oldest gene transfer techniques [2]. It is based on the interaction of positive charges on the DEAE-dextran molecule with the negatively charged backbone of nucleic acids. The DNA-DEAE-dextran complexes appear to adsorb onto cell surfaces and be taken up by endocytosis. 

Vallet-Regi, M. and Arcos D. (2005) Silicon substituted hydroxyapatites. A method to upgrade calcium phosphate based implants. Journal of Materials Chemistry, 15, 1509—1516. 

Macmillan and coworkers51 105 106 purified pectinesterase produced by Clostridium multifermentans by using practically all of the available methods and materials (calcium phosphate gel, DEAE-cellulose, DEAE-, QEAE-, CM-, and SE-Sephadex, Sephadexes G-75, G-100, G-150, and G-200, Sepharose 4B, and zonal centrifugation). However, they could not separate pectinesterase from exo-pectate lyase, and, hence, they postulated that either (a) a complex of the two enzymes having an apparent molecular weight of 400,000 exists, or (b) the two enzymes are identical in their molecular species. On the basis of the mode of action of this pectinesterase in comparison with that of those from tomatoes and from Fusarium ox-ysporum, the existence of a complex of pectinesterase and exopectate lyase in Clostridium multifermentans appears to be the more probable. 

A disadvantage of the conventional precipitation method in which the supersaturation was allowed to decrease during the reactions, was that different calcium phosphate phases could form and subsequently dissolve during the course of the reactions. In the present work, the constant composition method was used to investigate the influence of sodium chloride, potassium chloride, and potassium nitrate, as background electrolyte upon the rate of crystallization of HAP in solutions supersaturated only with respect to this phase. These experiments were made in solutions containing totaj... 

Transfection is the process of introducing DNA or RNA into eukaryotic ceils. The use of transfection is to study the role and regulation of proteins or to understand the mechanisms of a pathway. Transfection can be transient for rapid analysis or stable , mostly for induction of expression. There are various methods of transfection which include electroporation, viral vectors, DEAE-Dextran, calcium phosphate or Lipofectamine. The choice of transfection depends on the cell type used. The most desirable technique is the one which gives high efficiency of nucleic acid transfection with less interference to the cells physiology and high reproducibility. 

Again, there are several choices of extractant, and the preferred one depends mainly on the type of soil under test. One of the most widely used procedures is the Olsen method (Olsen ef al., 1954), which was developed in the USA to correlate crop response to fertilizer on calcareous soils. The amount of P extracted will vary with temperature (increases by 0.43 mg P kg- per degree rise between 20°C and 30°C) and shaking speed, so conditions should be standardized. The extractant is 0.5 M sodium bicarbonate adjusted to pH 8.5. The bicarbonate competes with phosphate on the adsorption sites extracts, and removes most, but not all of it, together with some soluble calcium phosphate. Addition of phosphate-free activated carbon before shaking is necessary if coloured soil extracts are obtained, and then they will require filtration. 

Table 11.2. Vector systems used to deliver genes into mammalian cells. To date, the majority of clinical trials undertaken have utilized retroviral vector systems. Non-viral systems have generally been employed least often, although some, e.g. nucleic acid-containing liposomes, may be used more extensively in the future. Some of the methods tested, e.g. calcium phosphate precipitation, electroporation and particle acceleration, are unlikely to be employed to any great extent in gene therapy protocols...

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