The authors thank Ernst Bamberg (Max-Planck-Institute of Biophysics, Frankfurt, Germany) for generous support during the initial phase of method development, Kazuhiro Abe (Kyoto University, Japan) for numerous fruitful discussions and Dr. Michael Kohl (Analytik-Service, Woltersdorf, Germany) for technical support. The authors gratefully acknowledge funding by the German Research Foundation DFG (Cluster of Excellence "Unifying Concepts in Catalysis"), which also financed the Perkin Elmer AAnalyst 800 apparatus (SFB 498).

1. cDNA Constructs and Protein Expression in Xenopus Oocytes
The cDNA of the membrane protein of interest should be cloned into a vector suitable for expression in Xenopus laevis oocytes such as pTLN9 or pcDNA3.1X10. Such optimized vectors contain the 5'- and 3'-untranslated regions (UTR) of the Xenopus laevis &beta;-globin gene flanking the multiple cloning site (MCS), an RNA polymerase promoter sequence (pTLN: SP6, pcDNA3.1X: T7) located before ...

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U.S.A. 108, 20556-20561 (2011).</li><li>Friedrich, T., Bamberg, E., Nagel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na+,K+-ATPase+pump+currents+in+giant+excised+patches+activated+by+an+ATP+concentration+jump.">Na+,K+-ATPase pump currents in giant excised patches activated by an ATP concentration jump.</a> Biophys J. 71, 2486-2500 (1996).</li><li>D&#252;rr, K. L., Tavraz, N. N., Dempski, R. E., Bamberg, E., Friedrich, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+significance+of+E2+state+stabilization+by+specific+alpha/beta-subunit+interactions+of+Na,K-+and++H,K-ATPase.">Functional significance of E2 state stabilization by specific alpha/beta-subunit interactions of Na,K- and H,K-ATPase.</a> J. Biol. Chem. 284, 3842-3854 (2009).</li><li>D&#252;rr, K. L., Abe, K., Tavraz, N. N., Friedrich, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E2P+state+stabilization+by+the+N-terminal+tail+of+the+H,K-ATPase+beta-subunit+is+critical+for+efficient+proton+pumping+under+in+vivo+conditions.">E2P state stabilization by the N-terminal tail of the H,K-ATPase beta-subunit is critical for efficient proton pumping under in vivo conditions.</a> J. Biol. Chem. 284, 20147-20154 (2009).</li><li>Durr, K. L., Tavraz, N. N., Zimmermann, D., Bamberg, E., Friedrich, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Na,K-ATPase+and+H,K-ATPase+enzymes+with+glycosylation-deficient+beta-subunit+variants+by+voltage-clamp+fluorometry+in+Xenopus+oocytes.">Characterization of Na,K-ATPase and H,K-ATPase enzymes with glycosylation-deficient beta-subunit variants by voltage-clamp fluorometry in Xenopus oocytes.</a> Biochemistry. 47, 4288-4297 (2008).</li><li>D&#252;rr, K. L., Tavraz, N. N., Friedrich, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+gastric+H,K-ATPase+activity+by+cations,+voltage+and+intracellular+pH+analyzed+by+voltage+clamp+fluorometry+in+Xenopus+oocytes.">Control of gastric H,K-ATPase activity by cations, voltage and intracellular pH analyzed by voltage clamp fluorometry in Xenopus oocytes.</a> PLoS One. 7, e33645 (2012).</li><li>Tavraz, N. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+functional+consequences+of+mutations+in+the+Na+/K+-ATPase+alpha2-subunit+causing+familial+hemiplegic+migraine+type+2.">Diverse functional consequences of mutations in the Na+/K+-ATPase alpha2-subunit causing familial hemiplegic migraine type 2.</a> J. Biol. Chem. 283, 31097-31106 (2008).</li><li>Lorenz, C., Pusch, M., Jentsch, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heteromultimeric+CLC+chloride+channels+with+novel+properties.">Heteromultimeric CLC chloride channels with novel properties.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 13362-13366 (1996).</li><li>Koenderink, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na,K-ATPase+mutations+in+familial+hemiplegic+migraine+lead+to+functional+inactivation.">Na,K-ATPase mutations in familial hemiplegic migraine lead to functional inactivation.</a> Biochim. Biophys. Acta. 1669, 61-68 (2005).</li><li>Price, E. M. J., Lingrel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+relationships+in+the+Na,K-ATPase+alpha+subunit:+site-directed+mutagenesis+of+glutamine-111+to+arginine+and+asparagine-122+to+aspartic+acid+generates+a+ouabain-resistant+enzyme.">Structure-function relationships in the Na,K-ATPase alpha subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme.</a> Biochemistry. 27, 8400-8408 (1988).</li><li>Richards, R., Dempski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+the+conformational+dynamics+of+membrane+proteins+in+situ+with+site-directed+fluorescence+labeling.">Examining the conformational dynamics of membrane proteins in situ with site-directed fluorescence labeling.</a> J. Vis. Exp. (2627), e2627 (2011).</li><li>Lafaire, A. V., Schwarz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+dependence+of+the+rheogenic+Na+/K++ATPase+in+the+membrane+of+oocytes+of+Xenopus+laevis.">Voltage dependence of the rheogenic Na+/K+ ATPase in the membrane of oocytes of Xenopus laevis.</a> J. Membr. Biol. 91, 43-51 (1986).</li><li>Morth, J. P., Pedersen, B. P., Toustrup-Jensen, M. S., Sorensen, T. L., Petersen, J., Andersen, J. P., Vilsen, B., Nissen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+sodium-potassium+pump.">Crystal structure of the sodium-potassium pump.</a> Nature. 450, 1043-1049 (2007).</li><li>Toustrup-Jensen, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C+terminus+of+Na+,K+-ATPase+controls+Na++affinity+on+both+sides+of+the+membrane+through+Arg935.">The C terminus of Na+,K+-ATPase controls Na+ affinity on both sides of the membrane through Arg935.</a> J. Biol. Chem. 284, 18715-18725 (2009).</li><li>Morth, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+Na+,K+-ATPase+and+mapping+of+isoform+differences+and+disease-related+mutations.">The structure of the Na+,K+-ATPase and mapping of isoform differences and disease-related mutations.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 364, 217-227 (2009).</li><li>Yaragatupalli, S., Olivera, J. F., Gatto, C., Artigas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+Na++transport+after+an+intracellular+alpha-subunit+deletion+reveals+strict+external+sequential+release+of+Na++from+the+Na/K+pump.">Altered Na+ transport after an intracellular alpha-subunit deletion reveals strict external sequential release of Na+ from the Na/K pump.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 15507-15512 (2009).</li><li>Meier, S., Tavraz, N. N., Durr, K. L., Friedrich, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperpolarization-activated+inward+leakage+currents+caused+by+deletion+or+mutation+of+carboxy-terminal+tyrosines+of+the+Na+/K+-ATPase+{alpha}+subunit.">Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase {alpha} subunit.</a> J. Gen. Physiol. 135, 115-134 (2010).</li><li>Vedovato, N., Gadsby, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+two+C-terminal+tyrosines+stabilize+occluded+Na/K+pump+conformations+containing+Na+or+K+ions.">The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions.</a> J. Gen. 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The described method to measure the amount of Rb+ (or Li+) taken up into individual Xenopus oocytes expressing Na+,K+- or H+,K+-ATPase has proven to be a versatile, flexible and accurate technique to determine the kinetic or thermodynamic parameters of transport for cation-countertransporting P-type ATPases1,4,5,7,8. It is a safe and reliable alternative to radioactive tracer flux assays, and allows addressing a large scope of experimen...

We wanted to develop a sensitive, safe and inexpensive alternative to radioactive tracer experiments to investigate the specific transport activity of ion translocating membrane proteins in order to circumvent restrictions regarding the access to isotope laboratories, safety requirements or the use of costly radioisotopes, which - as in the case of lithium - may even be unavailable due to extremely short decay times. We were particularly interested in determining the activity of the electroneutrally operating gastric H+,K+-ATPase, because the enzyme does not generate current and its activity can therefore not be addressed by electrophysiological methods. Since Na+,K+- and H+,K+-ATPase transport Rb+ as efficient as K+ (and Li+ as well), the high sensitivity of the AAS technique for rubidium or lithium should facilitate sensitive detection of transport activity. Atomic absorption spectrophotometers are common analytical devices, which are widely distributed in chemical laboratories and should be accessible to a large number of interested scientists. Furthermore, we wanted to take advantage of the Xenopus oocyte expression system, which utilizes large single cells (about 1.0-1.5 mm diameter) that allow to achieve a remarkably low cell-to-cell variability regarding the protein expression level within a single batch. A simple calculation demonstrates the feasibility of the AAS assay: The detection limit (characteristic mass) for rubidium with the THGA-AAS technique is 10 pg or 1.2&middot;10-13 mol (Rb: 85.47 g/mol), for lithium 5.5 pg or 7.9&middot;10-13 mol (Li: 6.94 g/mol). Upon heterologous expression of Na+,K+-ATPase in Xenopus oocytes, pump currents of 100 nA can be achieved (which equals about 6.2&middot;1011 elementary charges per second, or 1.03&middot;10-12 mol s-1), thus resulting in a transport of 6&middot;10-6 C of charge within 1 min. Since the transport of one net charge corresponds to the uptake of two Rb+ ions (due to the 3Na+/2K+ stoichiometry), 100 nA current for 1 min corresponds to an uptake of 1.2&middot;10-10 mol Rb+. Thus, even upon a 1,000-fold dilution (homogenization of an oocyte with about 1 &mu;l volume in 1 ml water), a typical THGA-AAS sample (20 &mu;l) contains 2.4&middot;10-12 mol Rb+ (or 204 pg), which is far above the detection threshold. Therefore, even transporters with more than 100-fold lower transport activity or plasma membrane expression can be assayed with the technique by appropriately adjusting the flux time of the experiment.
Since the pumping rate is sensitively dependent on temperature (typical activation energies for Na+,K+-ATPase are in the range of 90 kJ/mol to 130 kJ/mol1-3, which results in an about 30% increase in the turnover rate upon a change from 20 &deg;C to 22 &deg;C), it is mandatory to carry out the flux measurements under precise temperature control (air conditioning) with well equilibrated buffer solutions. Furthermore, oocytes should be carefully selected regarding homogenous size for the expression of an ion transporter. With these precautions, it is possible to routinely achieve experimental standard errors of less than 10 percent with about 10 cells per experimental condition. Using this technique, we were able to determine e.g. the apparent Rb+ affinities of cation transport4-6, the influence of extra- and intracellular pH7 and the effect of mutations of residues involved in cation coordination during transport4,8. An advantage of the technique is that ion fluxes can also be determined in combination with two-electrode voltage clamping of the oocytes, which on one hand assures accurate control of the membrane potential during transport and on the other hand allows to correlate ion flux with membrane current. Thus, it was possible to verify the 3Na+/2K+ stoichiometry of the Na+,K+-ATPase (see exemplary results below) and to determine the voltage dependence of cation transport of the gastric H+/K+-ATPase7.

<table border="1">
<tbody>
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 <td>Name 			of the Reagent</td>
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 <td>4.0 Ethicon Vicryl 			suture material</td>
 <td>Johnson &amp; 			Johnson</td>
 <td>V633H</td>
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 <td>Collagenase 			type 1A from Clostridium 			hystolyticum</td>
 <td>Sigma Aldrich</td>
 <td>C9891</td>
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 <td>High-Pure PCR Product Purification Kit</td>
 <td>Roche Applied Science</td>
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 <td>Nuclease-free water</td>
 <td>Ambion</td>
 <td>AM9937</td>
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 <td>mMessage mMachine Kit SP6/T7</td>
 <td>Ambion</td>
 <td>1340, 1344</td>
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 <td>Ouabain octahydrate</td>
 <td>Sigma Aldrich</td>
 <td>O3125</td>
 <td></td>
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 <td>Tricain (ethyl 3-aminobenzoate 			methanesulfonate salt)</td>
 <td>Sigma Aldrich</td>
 <td>A5040</td>
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 <td>Sigma Aldrich</td>
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 <td>SCH28080</td>
 <td>Sigma Aldrich</td>
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 <td>Hollow Cathode Lamp Lithium</td>
 <td>Photron</td>
 <td>P929LL</td>
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 <td>Hollow Cathode Lamp Rubidium</td>
 <td>Photron</td>
 <td> P945 </td>
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 <td>Micropipette Puller </td>
 <td>Narishige</td>
 <td>Model PC-10</td>
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 <td>Oocyte Recording Chamber RC-10</td>
 <td>Warner Instr.</td>
 <td>W4 64-0306</td>
 <td></td>
 </tr>
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 <td>Nanoject II Injection Pump</td>
 <td>Drummond Scientific</td>
 <td>3-000-204</td>
 <td></td>
 </tr>
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 <td>pCLAMP software</td>
 <td>Molecular Devices</td>
 <td></td>
 <td></td>
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 <td>Polypropylene Sample Cup 			(1.2 ml)</td>
 <td>Perkin Elmer</td>
 <td>B0510397</td>
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 <td>Speedvac - Concentrator model 5301</td>
 <td>Eppendorf</td>
 <td>5301 000.210</td>
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 <td>THGA Tube</td>
 <td>Perkin Elmer</td>
 <td>B3000641</td>
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 <td>Turbo TEC-10CX Amplifier</td>
 <td>NPI Electronics</td>
 <td>TEC-10CX</td>
 <td></td>
 </tr>
 </tbody>
</table>

measuring cation transport by na,k- and h,k-atpase in xenopus oocytes by atomic absorption spectrophotometry: an alternative to radioisotope assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Quantification of Rb+ uptake by K+- (or Rb+)-countertransporting P-type ATPases by AAS in the Xenopus oocyte expression system permits reliable determination of enzyme kinetic parameters.
Determination of the transport stoichiometry of the Na+/K+-ATPase
For the electrogenic Na+/K+-ATPase, Rb+ fluxes can be determined in two-electrode voltage-clamp experiments aimed at the c...

Watch this Scientific Journal Video about Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays at JoVE.co

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

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10.4K ビュー.  Technical University of Berlin. We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

ビデオ: Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic...

atomic absorbance spectroscopy to measure intracellular zinc pools in mammalian cells

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance...

determining na+-k+-atpase and ca2+-mg2+-atpase activity in salidroside treated mcf-7 cells

Determining Na+-K+-ATPase and Ca2+-Mg2+-ATPase Activity in Salidroside Treated MCF-7 Cells

contribution of the na+/k+ pump to rhythmic bursting, explored with modeling and dynamic clamp analyses

Contribution of the Na+/K+ Pump to Rhythmic Bursting, Explored with Modeling and Dynamic Clamp Analyses

Presented here is a method for investigation of the roles of the Na+/K+ pump and persistent Na+ current in leech heart interneurons using dynamic...

Contribution of the Na+/K+ Pump to Rhythmic Bursting, Explored with Modeling and Dynamic Clamp Analyses

manipulation and in vitro maturation of xenopus laevis oocytes, followed by intracytoplasmic sperm injection, to study embryonic development

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic...

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

measuring trans-plasma membrane electron transport by c2c12 myotubes

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

The goal of this protocol is to spectrophotometrically monitor trans-plasma membrane electron transport utilizing extracellular electron acceptors and to analyze enzymatic interactions that may occur with these extracellular electron...

study of the functions and activities of neuronal k-cl co-transporter kcc2 using western blotting

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting

The present protocol highlights the application of western blotting technique to study the functions and activities of neuronal K-Cl co-transporter KCC2. The protocol describes the investigation of KCC2 phosphorylation at kinase regulatory sites Thr906/1007 via western blotting. Also, additional methods to confirm KCC2 activity are briefly highlighted in this...

a robust pneumonia model in immunocompetent rodents to evaluate antibacterial efficacy against s. pneumoniae, h. influenzae, k. pneumoniae, p. aeruginosa or a. baumannii

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

visualizing rna localization in xenopus oocytes

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal...

Visualizing RNA Localization in Xenopus Oocytes

measuring in vitro atpase activity for enzymatic characterization

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified...

Measuring In Vitro ATPase Activity for Enzymatic Characterization

microinjection of xenopus laevis oocytes

Microinjection of Xenopus Laevis Oocytes

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.<br...

use of electron paramagnetic resonance in biological samples at ambient temperature and 77 k

Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K

Electron paramagnetic resonance (EPR) spectroscopy is an unambiguous method to measure free radicals. The use of selective spin probes allows for detection of free radicals in different cellular compartments. We present a practical, efficient method to collect biological samples that facilitate treating, storing, and transferring samples for EPR...

recording gap junction current from xenopus oocytes

Recording Gap Junction Current from Xenopus Oocytes

Here we present a protocol to express gap junction proteins in Xenopus oocytes and record junctional current between two apposed oocytes using a commercial amplifier designed for dual oocyte voltage-clamp recordings in a high side current measuring...

Recording Gap Junction Current from Xenopus Oocytes

simultaneous quantification of t-cell receptor excision circles (trecs) and k-deleting recombination excision circles (krecs) by real-time pcr

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow...

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

enrichment of mammalian tissues and xenopus oocytes with cholesterol

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ...

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

voltage-clamp fluorometry in xenopus oocytes using fluorescent unnatural amino acids

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence...

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

functional characterization of na+/h+ exchangers of intracellular compartments using proton-killing selection to express them at the plasma membrane

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

heterologous expression and functional analysis of aedes aegypti odorant receptors to human odors in xenopus oocytes

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes

A protocol is presented that functionally characterizes mosquito ORs in response to human odors using a Xenopus oocyte expression system coupled with a two-electrode voltage clamp, providing a powerful new technique for exploring the responses of mosquitoes ORs to exposure to human...

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes

meiotic spindle assessment in mouse oocytes by sirna-mediated silencing

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism 2, 3. Auxin activates a H+ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins 4, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins 5. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.
 To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 &deg;C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...