प्राथमिक संवर्धित hippocampal न्यूरॉन्स में घने कोर vesicles के लाइव इमेजिंग

Summary

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.

Abstract

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.

Protocol

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?…

Discussion

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?…

Materials

Material Name	Tipo	Company	Catalogue Number	Comment
MEM-Eagle with Earle salts and L-glutamine	Reagent	Mediatech	10-010-CV
Lipofectamine 2000	Reagent	Invitrogen	11668-027	Transfection reagent
10X Hanks with Ca2+ and Mg2+	Reagent	Gibco/Invitrogen	14185-052	Live-imaging medium
1M HEPES	Reagent	Gibco/Invitrogen	15630-130	Live-imaging medium