Name: tissue collection of bats for -omics analyses and primary cell culture
Uploaded: 2019-10-23T13:00:00
Description: Watch this Scientific Journal Video about Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture at JoVE.com

हम सेंट्रो डे इकोलॉजी वाई बायोडाइडिसिडैड CEBIO, एरिका पैलिज़ा, मिलुस्का सांचेज़, जॉर्ज कैरेरा, एडगर रेंगिफो वीज़, हेरोल्ड पोरोकैरेरो ज़रिया, जॉर्ज रुज़ लेवेउ, जैमे पेचेको कैस्टिलो, कार्लोस टेलो, फैनी कोर्नेजो, और फैनी फर्नान्डेज को धन्यवाद देते हैं। पेरू में संग्रह संभव. हम भी डोमिनिकन गणराज्य में ऊतक संग्रह संभव बनाने के लिए Grupo Jaragua और Yolanda Len के सभी सदस्यों को धन्यवाद, साथ ही Bernal Rodriguez Hernandez, Bernal Matarrita, और कोस्टा रिका में ला सेल्वा जैविक अनुसंधान स्टेशन में हर कोई बनाने के लिए, नमूना संभव है. हम सेल संस्कृति पीढ़ी के लिए Phyllostomus discolor चमगादड़ से उपयोग और पंख घूंसे के नमूना संग्रह के लिए एला Lattenkamp और Lutz Wiegrebe धन्यवाद. Phyllostomus discolor चमगादड़ म्यूनिख में लुडविग-मैक्सिमिलियन्स विश्वविद्यालय के विभाग जीव विज्ञान द्वितीय में एक प्रजनन कॉलोनी से उत्पन्न. म्यूनिख जिला पशु चिकित्सा कार्यालय द्वारा चमगादड़ों को रखने और प्रजनन करने की स्वीकृति जारी की गई थी। LMD, SJR, KTJD, और LRY NSF-डीईबी 1442142 द्वारा वित्त पोषित किया गया. एलएमडी NSF-DEB 1838273 द्वारा वित्त पोषित किया गया था. एलआरवाई को एनएसएफ-पीआरएफबी 1812035 द्वारा वित्त पोषित किया गया था। SCV और पीडी एक मैक्स प्लैंक अनुसंधान समूह पुरस्कार, और एक मानव फ्रंटियर्स विज्ञान कार्यक्रम (HFSP) अनुसंधान अनुदान (RGP0058/2016) द्वारा वित्त पोषित किया गया. SJR, JHTP, और KTJD यूरोपीय अनुसंधान परिषद द्वारा वित्त पोषित किया गया (ईआरसी शुरू अनुदान 310482 [EVOGENO]) SJR को सम्मानित किया, ईसीटी यूरोपीय अनुसंधान परिषद अनुदान (ईआरसी-2012-StG311000) द्वारा वित्त पोषित किया गया था।

यहाँ वर्णित सभी तरीकों को Stony Brook विश्वविद्यालय के संस्थागत पशु देखभाल और उपयोग समिति (IACUC) द्वारा अनुमोदित किया गया है (प्रोटोकॉल #2013-2034).
1. इच्छामृत्यु
<ol><li>एक isoflurane संवेदनाहारी या किसी अन्य उपलब्ध स...

इस पांडुलिपि में चर्चा किए गए प्रोटोकॉल में चमगादड़ों के विभिन्न उच्च-थ्रूपुट आण्विक विश्लेषणों के लिए सर्वोत्तम नमूना पद्धतियों का वर्णन किया गया है। सभी सफल -omics अध्ययन उच्च गुणवत्ता ऊतक की आवश्यकत?...

<table>
	<tbody>
		<tr>
			<td>1.5 mL Eppendorf Safelock tubes</td>
			<td>Fischer Scientific</td>
			<td>10509691</td>
			<td>1.5 mL sterile tubes used during cell culture protocol</td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Sarstedt</td>
			<td>62,554,002</td>
			<td>15 mL sterile tubes used during cell culture protocol</td>
		</tr>
		<tr>
			<td>2 mL cryovials</td>
			<td>Thomas Scientific</td>
			<td>1154P75</td>
			<td>cryogenic vials for tissues to be stored in liquid nitrogen</td>
		</tr>
		<tr>
			<td>3-mm biopsy punch</td>
			<td>Medline</td>
			<td>MIL3332</td>
			<td>wing biopsy punch for cell culture</td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Greiner</td>
			<td>83,392</td>
			<td>Culture vessle</td>
		</tr>
		<tr>
			<td>Allprotect Tissue Reagent</td>
			<td>Qiagen</td>
			<td>76405</td>
			<td>for fecal samples; tissue stabilizing solution</td>
		</tr>
		<tr>
			<td>Cell counting slides for TC10/TC20 cell counter, dual chamber</td>
			<td>Bio-Rad</td>
			<td>145-0011</td>
			<td>Chambers for the count cells using the automated cell counter TC20 by Bio-Rad</td>
		</tr>
		<tr>
			<td>collagenase IV</td>
			<td>Stemcell Technologies</td>
			<td>7909</td>
			<td>For dissociation of primary cells</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>D2650-5X5ML</td>
			<td>Prevents crystalization of water during freezing of the cells.</td>
		</tr>
		<tr>
			<td>DMEM&#8211;Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Thermo Fisher</td>
			<td>12491-015</td>
			<td>culture media</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS</td>
			<td>Invitrogen</td>
			<td>14190169</td>
			<td>Balanced salt solution used for washing cells</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Fischer Scientific</td>
			<td>10270106</td>
			<td>Serum-supplement for in vitro cell culture of eukaryotic cells</td>
		</tr>
		<tr>
			<td>Gentamycin sulfate salt</td>
			<td>Sigma Aldrich</td>
			<td>G1264-250MG</td>
			<td>Antibiotic for culture media</td>
		</tr>
		<tr>
			<td>Nalgene Mr. Frosty</td>
			<td>Thermo Scientific</td>
			<td>5100-0050</td>
			<td>Freezing container which provides 1&#176;C/min cooling rate</td>
		</tr>
		<tr>
			<td>PARAFILM</td>
			<td>Sigma</td>
			<td>P7793</td>
			<td>Wrapping tubes etc for sealing</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (pen-strep), 100x</td>
			<td>Invitrogen</td>
			<td>15140130</td>
			<td>Antibiotic for culture media</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS) 10x, pH 7.4</td>
			<td>Thermo Fisher</td>
			<td>10010023</td>
			<td>salt buffer used for washes and storage of bone tissue; dilute to 10X using de-ionized water</td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>Thermo Fisher</td>
			<td>AM7021</td>
			<td>RNA stabilizing solution</td>
		</tr>
		<tr>
			<td>RNAse away</td>
			<td>Genetech</td>
			<td>83931-250mL</td>
			<td>breaks down enzymes that lead to RNA degradation</td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>Fisher Scientific</td>
			<td>7631-86-9</td>
			<td>dessicant agent</td>
		</tr>
		<tr>
			<td>TC20 Automated Cell Counter</td>
			<td>Bio-Rad</td>
			<td>1450102</td>
			<td>Automated cell counter</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Bio-Rad</td>
			<td>145-0013</td>
			<td>Cell stain used to assess cell viability</td>
		</tr>
		<tr>
			<td>trypsin-EDTA</td>
			<td>Sigma Aldrich</td>
			<td>T4049</td>
			<td>For dissociation of cells during splitting</td>
		</tr>
	</tbody>
</table>

tissue collection of bats for -omics analyses and primary cell culture

उच्च थ्रूपुट अनुक्रमण प्रौद्योगिकियों अग्रिम के रूप में, उच्च गुणवत्ता वाले ऊतक अधिग्रहण और संरक्षण के लिए मानकीकृत तरीकों गैर मॉडल जीवों के लिए इन तरीकों के विस्तार के लिए अनुमति देते हैं। चमगादड़ से ऊतक संग्रह का अनुकूलन करने के लिए प्रोटोकॉल की एक श्रृंखला उच्च थ्रूपुट अनुक्रमण दृष्टिकोण की एक श्रृंखला के लिए विकसित किया गया है। यहाँ रेखांकित चमगादड़, वांछित जनसांख्यिकी प्रत्येक बल्ले के लिए एकत्र किया जा करने के लिए, और अनुकूलित तरीकों ऊतक संग्रह के दौरान एक बल्ले पर तनाव को कम करने के लिए कब्जा करने के लिए प्रोटोकॉल हैं। विशेष रूप से रेखांकित ऊतक को प्राप्त करने के लिए (i) उच्च आणविक वजन जीनोमिक विश्लेषण के लिए डीएनए, (ii) ऊतक-विशिष्ट ट्रांसक्रिप्टम के लिए आरएनए, और (iii) प्रोटीओमिक स्तर के विश्लेषण के लिए प्रोटीन प्राप्त करने के लिए तरीके हैं। अंत में, यह भी रेखांकित विंग क्लिप से व्यवहार्य प्राथमिक सेल संस्कृतियों बनाने के द्वारा घातक नमूना से बचने के लिए एक तरीका है. इन तरीकों की एक केंद्रीय प्रेरणा प्रत्येक बल्ले के लिए संभावित आणविक और रूपात्मक डेटा की मात्रा को अधिकतम करने और ऊतकों को संरक्षित करने के लिए इष्टतम तरीके सुझाने के लिए है ताकि वे भविष्य में विकसित नए तरीकों के रूप में अपने मूल्य को बनाए रखें। यह मानकीकरण विशेष रूप से महत्वपूर्ण हो गया है क्योंकि क्रोमोसोम स्तर, दुनिया भर में प्रजातियों के त्रुटि-मुक्त जीनोम के अनुक्रम के लिए पहल उभरा है, जिसमें कई वैज्ञानिक दलों विभिन्न वर्गीकरण के अनुक्रमण का नेतृत्व कर रहे हैं समूहों. प्रोटोकॉल यहाँ उल्लिखित आदर्श ऊतक संग्रह और Bat1K के लिए ऊतक संरक्षण विधियों को परिभाषित, संघ है कि बल्ले की हर प्रजाति के जीनोम अनुक्रमण है.

डीएनए मानक कम आणविक वजन (LMW) विश्लेषण के लिए, डीएनए तीन netropical चमगादड़ प्रजातियों से निकाला गया था. ऊतक नमूने डोमिनिकन गणराज्य और कोस्टा रिका से क्षेत्र में एकत्र किए गए थे, इस पत्र में वर...

Watch this Scientific Journal Video about Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture at JoVE.com

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

यह जीनोमिक, ट्रांसक्रिप्टोमिक, और चमगादड़ के प्रोटीओमिक विश्लेषण के लिए इष्टतम ऊतक तैयार करने के लिए एक प्रोटोकॉल है जो जंगली में पकड़ा गया है। यह बल्ले पर कब्जा और विच्छेदन, ऊतक संरक्षण, और बल्ले ऊतक के सेल culturing के लिए प्रोटोकॉल भी शामिल है.

-Omics विश्लेषण और प्राथमिक सेल संस्कृति के लिए चमगादड़ के ऊतक संग्रह

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

tissue-collection-of-bats-for-omics-analyses-and-primary-cell-culture

Research

JoVE Journal

Biology

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

एफ actin के लिए बाइंडिंग प्रोटीन का विश्लेषण के लिए, actin सह अवसादन परख

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

ऊतक इंजीनियरिंग और सेल संस्कृति के लिए रेशेदार पॉलिमर scaffolds Electrospinning

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

प्रेरण और सेल संस्कृति में Hypoxia का परीक्षण

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

अलगाव और चूहा यकृत कोशिकाओं की प्राथमिक संस्कृति

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies.

Ultrastructural विश्लेषण के लिए खमीर endosomal प्रणाली की Nanogold लेबल

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

प्राथमिक आंतों Organoid संस्कृति में रेट्रोवायरल संक्रमण का एक वीडियो प्रोटोकॉल

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

अलगाव और माउस महाधमनी endothelial कोशिकाओं के प्राथमिक संस्कृति

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

Immunohistochemical और Immunofluorescent विश्लेषण के लिए आंतों के ऊतकों तैयारी के लिए स्विस रोलिंग तकनीक में सुधार

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

एस्ट्रोजन का उत्पादन प्राथमिक गोजातीय Granulosa कोशिकाओं के एक ऊतक संस्कृति मॉडल

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

चूहा adrenocortical कोशिकाओं और steroidogenic कार्यों के assays की प्राथमिक संस्कृति

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...