This study optimized the protocol of calcium phosphate transfection for mscs and highlighted the importance of serum during the process of transfection. Calcium phosphate magnetic nanoparticles have been successfully developed by puddu et al. Waisman center, university of wisconsinmadison, madison, wi. This technique has been used to obtain both transient 1 and stable 2 transfections in a wide variety of cell types. Aug 05, 2016 calcium phosphate based transfection is a standard and well known technique. This unit contains two methods of calcium phosphate based eukaryotic cell transfection, protocols that can be used for both transient and stable transfections. Calcium phosphate transfection kingston 1999 current. At the time of transfection, cells should be about 3040% confluent. Sep 17, 2019 calcium phosphate transfection is an inexpensive and welldescribed method without discernible biosafety issues. Principle dna is mixed with calcium chloride addition to buffered saline phosphate solution and incubating at room temperature formation of dna calcium phosphate coprecipitates which adhere to surface of cells. Amounts refer to a transfection of one p100 of cells 1 precipitate 1020 g of dna.
Materials for calcium phosphate transfection hela cells complete dmem dna 10 50 ug per transfection 2. Calcium phosphate transfection kingston 2003 current. The critical factor in capo 4 coprecipitation transfections is the ph of the solutions. This class of methods uses chemicals such as lipids and calcium phosphate for transfection. The critical factor in capo4 coprecipitation transfections is the ph of the solutions. Twentyfour hours prior to transfection, inoculate 1 to 2 x106 cells10 cm plate or, inoculate 3x106 cellsplate 16hours prior to transfection. Calcium phosphate transfection thermo fisher scientific. At the time of transfection, cells should be about 3040%. Upon oral uptake, calcium phosphate competes for and blocks the absorption of radium ra226 and strontium sr90 in the gastrointestinal gi tract. The uptake and stability of simianvirus40 dna after calcium phosphate transfection of cv1 cells. The precipitate is taken up by the cells via endocytosis or phagocytosis. The protocol is versatile and, although used primarily on adherent cells in culture, also can be modified to transfect cells growing in suspension 10. The procedure is based on slow mixing of hepesbuffered saline containing sodium phosphate with a cacl 2 solution containing the dna. Calcium phosphate transfection thaler center for hiv and.
Some cells are calcium tolerant and can live in the presence of ca overnight, while other cells are calcium intolerant and should be exposed to the precipitate for no longer than 46 hours. Calcium phosphate transfection kit protocol sigmaaldrich. Highly effective precipitates for transfection purposes, however, can be generated only in a very narrow range of physicochemical conditions that control the initiation and growth of precipitate complexes. Transfection of adherent and suspended cells by calcium. Early transfection studies used viral rna and dna 2, 4, which, at that time, were relatively easy to propagate and purify, and allowed. This method also works well for transient expression of transfected genes and can be used with most adherent cell lines. High efficiency production of lentiviral vectors in hek. Calcium phosphate an overview sciencedirect topics. Calcium phosphate transfection is one of the chemical transfection methods using this principle.
For those experiments where more transfection mix is needed, simply use a multiple of the reagents described below. Calcium phosphate transfection current protocols wiley. Calcium phosphate transfection optimization for serumfree. Principle dna is mixed with calcium chloride addition to buffered salinephosphate solution and incubating at room temperature formation of dnacalcium phosphate coprecipitates which adhere to surface of cells. Difference between transfection and transformation. In our study on calcium phosphate transfection, we used both ethidium monoazide and pna labelling and we analyzed plasmid uptake in individual cells. Transient assays for gene expression in transfected cells are performed 4872hrs post transfection. Calcium phosphate transfection is a commonly used method for the introduction of dna into eukaryotic cells. Calcium phosphate transfection of packaging cells quantities for one 15cm plate. Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. Calcium divalent ions bind the dna polymers and neutralize their negatively charged phosphate backbones. This unit contains two methods of calcium phosphate. Pdf transfection system of aminofunctionalized calcium.
High efficiency production of lentiviral vectors in hek 293ft cells using plasmid co transfection. Transfection guide overview of transfection methods. Calcium phosphate transfection is routinely used for both transient and stable transfection of a variety of cell types. Lipidmediated transfection, also known as lipofection, uses lipids with similar properties to that of cell membrane.
The calcium phosphate transfection is an inexpensive and simple. The procedure is based on slow mixing of hepesbuffered saline containing sodium phosphate with a cacl 2 solution. Protocol for lentivial vector lv production 2nd generation. Depending on the time of assay, plate sparsely or more densely. Calcium phosphate transfection of 3t3 fibroblasts csh protocols. Calcium phosphate transfection is one of the most commonly used methods to deliver plasmid dna into mammalian cells.
A reduction of the calcium concentration from 250 mm to 100 mm, yielded a 25fold increase in the expression of the recombinant protein compared to the serumfree standard conditions. Aug 05, 2016 calcium phosphate transient transfection protocol. Lenti virus can be packaged by any transfection reagent. However, rapid particle growth has largely prevented the translation of this method for in vivo purposes. However, calcium phosphate coprecipitation is prone to variability and is not suited for in vivo gene transfer into whole animals.
With optimization, it is possible to use this method on hardtotransfect cells like primary neurons. Calcium phosphatemediated transfection of eukaryotic. Briefly, lentivirus was packaged by co transfection of larlbgfp construct with the packaging and envelope plasmids pspax2 and pmd2. The imfectin method requires a small amount of plasmid dnas and is simpleefficient. Calcium phosphate transfection generates mammalian. This technique involves mixing your dna construct with calcium chloride in a buffered saline phosphate solution. Drosophila schneider 2 s2 cells can be transfected with the recombinant expression vector alone for transient expression studies or in combination with a selection vector e. May 29, 2008 transfection with polyethylenimine pei was evaluated as a method for the generation of recombinant chinese hamster ovary cho dg44 cell lines by direct comparison with calcium phosphate. Transfectopedia methods transit certified transfection. The ph should be checked with nonbleeding sterile ph sticks or reliable ph meters. We developed quantitative flow cytometry to analyze the correlations between dna uptake and other parameters known to influence transfection efficiency exposure time to plasmidcapi. Calcium phosphate transfection is the method of choice to produce longterm stable transfectants. Calcium phosphate transfection of adherent cells reagents 2x hbs 280mm nacl 8. The positively charged lipids associate with the negatively charged phosphate groups of genetic materials.
Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. Surface functionalization of cap nanoparticles cap nps with n2aminoethyl3aminopropyltrimethoxysilane was shown recently to. High efficiency production of lentiviral vectors in hek 293ft. Calcium phosphate transfection works very well for 293 cells up to 95% transfection efficiencies. In the protocols, plasmid dna is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. Calcium phosphatemediated dna transfection springerlink. Transfection with polyethylenimine pei was evaluated as a method for the generation of recombinant chinese hamster ovary cho dg44 cell lines by direct comparison with calcium phosphate. Calcium phosphate biomaterials are well known in the medical materials and devices world, in particular, for their use in the treatment of osseous defects and as coatings on metal alloy.
This can be achieved with compounds like calcium phosphate, polycations, and liposomes or more current technologies such as cationic lipids, polymers, dendrimers, and nanoparticles. The calcium phosphate transfection method for introducing dna into mammalian cells is based on forming a calcium phosphate dna precipitate. Calcium phosphate transfection of eukaryotic cells. This protocol is a modified version of a published method4, in which calcium phosphate based transfection methods for chinese hamster ovary cells and the 293 line of. Dna coprecipitation capo4 using both green fluorescent protein gfp and a monoclonal antibody as reporter proteins. Detailed instructions for the transfer of the rphyco erythrin protein into. Product components and storage conditions product cat. Mixing dna with calcium chloride in a phosphate buffer. The calcium phosphate transfection protocol is routinely used for both transient and stable transfection of a variety of cell types. In these protocols, plasmid dna is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. G by calcium phosphate transfection into hek293t cells. Dna calcium phosphate coprecipitates arise spontaneously in supersaturated solutions. Factors that can be optimized for calcium phosphate transfections are presented in.
C and 5% co 2 and split 110 every few days so they do not become over confluent. Application calcium phosphate transfection kit has been used. Utilize the following mix components for all experiments. Calcium phosphate and deaedextran reagents were incorporated into the first chemical methods that successfully transferred nucleic acid directly to cultured mammalian cells in a process referred to as transfection 1, 2, 3, 4. The calcium phosphate transfection method for introducing dna into mammalian cells is based on forming. This protocol has been optimized for transfection of neonatal rat cardiac myocytes. Chemical transfection transfections using chemical transfection reagents rely on electrostatic interactions to bind with nucleic acids and to target cell membranes. The following protocol is written for transfection of cells in a 10cm. Many methods have been developed in order to use calcium phosphate cap for delivering nucleotides into living cells. Mechanisms underlying this transfection method are not yet defined. The most common transfection methods are chemicalmediated transfection such as calcium phosphate coprecipitation, liposomes, and physical methods such as electroporation, gene gun technique, and microinjection. Calcium phosphate transient transfection protocol and guide. Calcium phosphate transfection has been optimized and widely used with.
Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection. Calcium phosphate transfection is wellestablished and safe, but the protocols need to be optimized according to different cell types. Calcium phosphate is the calcium salt of phosphoric acid with widely used applications. For those experiments where more transfection mix is. Transfect as soon as they are adherent and 7585% confluent. Calcium phosphate transfection of primary hippocampal neurons. Transfer of high copy number plasmid into mammalian cells. In animal cells, transfection is the preferred term. This technique has been used to obtain both transient and stable transfections in a wide variety of cell types. The calcium phosphate transfection is a widely used method for introducing foreign dna plasmids into cells.
Calcium phosphate transfection of 293t cells 2 transfection 1. Calcium phosphate transfection of primary hippocampal. Currently, there is no optimized protocol for mscs. Optimization and characterization of calcium phosphate. Calcium phosphatetransfection kit thermo fisher scientific. This step generates a precipitate that is dispersed onto the cultured cells. This method relies on the use of positively charged calcium ions to bind to the negatively charged phosphate backbone of dna and form a coprecipitation complex that can then be added to. Whether such microprecipitates are formed and whether they are relevant to the transfection remain open questions.
It may also refer to other methods and cell types, although other terms are often preferred. Transfection can result in unexpected morphologies and abnormalities in target cells. When the mixture is incubated at room temperature, dna calcium phosphate coprecipitates form. All solutions in the fivephoton biochemicals calcium phosphate transfection kits are provided sterile and adjusted to ph 7.
For cells on 24 well plates, combine equal amounts of the plasmid in question and normalization signal with l7rhbetagal plasmid. For cells on 24 well plates, combine equal amounts of the plasmid in question and normalization signal with l7rhbeta. The uptake and fate of dna transfected into mammalian cells in vitro. This unit presents two methods of calcium phosphate.
Transfer of high copy number plasmid into mammalian cells by. Calcium phosphate based transfection is a standard and well known technique. The dna to be expressed is cloned into the pmt2t or related vector which contains a sv40 origin, adeno major late promoter, the adeno tripartite leader and the va rna i gene, leading to very high level expression of the recombinant protein. Supplied reagents are sufficient to perform 1 control and 75 transfection reactions. Drosophila schneider 2 s2 cells thermo fisher scientific. Calcium phosphate facilitates the binding of the dna to the cell surface. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.
Calcium phosphate transfection protocol utilize the following mix components for all experiments. Transfection system of aminofunctionalized calcium phosphate. Since the solutions are of low ionic strength, often 2 or 3 ph meters must be consulted to correctly determine the ph. Due to its highly consistent ph and salt concentration, our calphos calcium phosphate transfection kit gives you the consistent transfection efficiency that you cannot always achieve with home brew versions. Calcium phosphate transfection of mammalian cultured cells. Calcium phosphate cap is the main mineral found in human bone and teeth and is considered as a highly biocompatible inorganic biomaterial legeros, 2008. Surface functionalization of cap nanoparticles cap nps with n2aminoethyl3aminopropyltrimethoxysilane was shown recently to achieve dispersed nps with a positive surface charge, capable of transfection chem. For those experiments where more transfection mix is needed, simply use a multiple of the reagents.
Pdf calcium phosphate transfection generates mammalian. Calcium phosphate transfection kit most cost effective. Nonadherent cell lines can be transfected by electroporation and liposome mediated transfection. This agent can be used as a countermeasure for exposure to strontium and radium radionuclides. Calcium phosphate transfection operates effectively in hek cells, which is the most commonly used cell culture transfection model. Both methods of transfection require very highquality plasmid.
Transfection can be carried out using calcium phosphate i. In this report, we optimized the protocol of calcium phosphate transfection for murine mscs, and compared this protocol with other gene transfer methods in different. Transfection at 70% confluency is suitable for both transfection methods. Using standard phosphate and calcium concentrations for transfection in the absence of serum resulted in titers of only 1% of those observed in the presence of serum.
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