Name: live imaging of dense-core vesicles in primary cultured hippocampal neurons
Uploaded: 2009-05-29T00:00:00
Description: Watch this Scientific Journal Video about Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons at JoVE.com

हम उनके इस पांडुलिपि के सावधान पढ़ने के लिए Harald Hutter और हेलेना डेकर धन्यवाद. हम भी अपनी तकनीकी विशेषज्ञता के लिए Leica माइक्रोसिस्टम्स से रेग सिद्धू धन्यवाद. इस शोध राष्ट्रीय विज्ञान और इंजीनियरिंग परिषद, कनाडा के 327100-06 # पुरस्कार, और साइमन फ्रेजर विश्वविद्यालय विज्ञान के संकाय द्वारा समर्थित किया गया.

भाग 1: 2000 Lipofectamine (Invitrogen) का उपयोग न्यूरॉन्स के अभिकर्मक सेट - अप उपकरण: चूहा या माउस hippocampal न्यूरॉन्स Kaech और बैंकर, 2006 के अनुसार सुसंस्कृत हैं. [4] ठेठ सेल transfections के लिए इस्तेमाल किया घनत्व 250,000 cells/6-cm पक?...

<ol>
	<li>Kulic, I. M., 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+role+of+microtubule+movement+in+bidirectional+organelle+transport.">The role of microtubule movement in bidirectional organelle transport.</a> Proc Natl Acad Sci U S A. 105 (29), 10011-10016 (2008).</li><li>Jacobson, C., Schnapp, B., Banker, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+change+in+the+selective+translocation+of+the+Kinesin-1+motor+domain+marks+the+initial+specification+of+the+axon.">A change in the selective translocation of the Kinesin-1 motor domain marks the initial specification of the axon.</a> Neuron. 49 (6), 797-804 (2006).</li><li>Barkus, R. V., 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=Identification+of+an+Axonal+Kinesin-3+Motor+for+Fast+Anterograde+Vesicle+Transport+that+Facilitates+Retrograde+Transport+of+Neuropeptides.">Identification of an Axonal Kinesin-3 Motor for Fast Anterograde Vesicle Transport that Facilitates Retrograde Transport of Neuropeptides.</a> Mol Biol Cell. 19 (1), 274-283 (2008).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat Protoc. 1 (5), 2406-2415 (2006).</li></ol>

लाइव सेल इमेजिंग organelle सभ्य न्यूरॉन्स में परिवहन के प्रत्यक्ष प्रेक्षण के लिए एक चुनौती है, लेकिन शक्तिशाली तकनीक है. कठिनाइयाँ अपस्ट्रीम गरीब न्यूरॉन संस्कृति जटिलताओं की वजह से स्वास्थ्य के साथ प्र?...

A correction was made to <a href="http://www.jove.com/details.php?ID=1144">Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons</a>. There was an error in the author's name. The author name was corrected to include a middle initial as follows:

David M. Kwinter, Michael A. Silverman

instead of:

David Kwinter, Michael Silverman.

A correction to author names has been made for article Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons.

Erratum: Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons.

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<td>MEM-Eagle with Earle salts and L-glutamine</td>
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<td>10X Hanks with Ca2+ and Mg2+</td>
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<td>1M HEPES</td>
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live imaging of dense-core vesicles in primary cultured hippocampal neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Watch this Scientific Journal Video about Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons at JoVE.com

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

लाइव सेल इमेजिंग विशेष उपयोगिता है जब organelle तस्करी की गतिशीलता का अध्ययन. यहाँ हम व्यापक क्षेत्र प्रतिदीप्ति माइक्रोस्कोपी का उपयोग सभ्य न्यूरॉन्स में घने कोर vesicles के रहते इमेजिंग के लिए एक प्रोटोकॉल का वर्णन. इस प्रोटोकॉल लचीला है और ऐसे mitochondria के रूप में में छवि अन्य organelles के लिए अनुकूलित किया जा सकता है, endosomes, और peroxisomes.

प्राथमिक संवर्धित hippocampal न्यूरॉन्स में घने कोर vesicles के लाइव इमेजिंग

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Research

JoVE Journal

Biology

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P0 नवजात चूहे से hippocampal न्यूरॉन्स के प्राथमिक संस्कृति

The physiological properties of hippocampal neurons are commonly investigated, especially because of the involvement of the hippocampus in learning and ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

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The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

Adenovirus की मध्यस्थता संवर्धित प्राथमिक माउस भ्रूणीय Fibroblasts में संकेतन अणुओं के आनुवंशिक हटाना

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

स्टोर संचालित जीवित कोशिकाओं में कैल्शियम प्रविष्टि प्रतिदीप्ति आधारित मापन संवर्धित सेल कैंसर से कंकाल की मांसपेशी फाइबर

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

4D Imaging of Protein Aggregation in Live Cells

4D जीवित कोशिकाओं में प्रोटीन एकत्रीकरण की इमेजिंग

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

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Apoptotic सेल क्लीयरेंस की इमेजिंग के दौरान जीते<em&gt; ड्रोसोफिला</em&gt; Embryogenesis

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

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संवर्धित में organelle परिवहन<em&gt; ड्रोसोफिला</em&gt; सेल: S2 सेल लाइन और प्राथमिक न्यूरॉन्स.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

लाइव प्राथमिक कोशिकाओं में G3BP तनाव granules गतिशीलता के दृश्य

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly ...

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

प्राथमिक चूहा नवजात cardiomyocytes लाइव सेल इमेजिंग के adenoviral के बाद और Lentiviral पारगमन Confocal डिस्क माइक्रोस्कोपी स्पिनिंग का प्रयोग

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...