Name: isolation of small noncoding rnas from human serum
Uploaded: 2014-06-19T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Small Noncoding RNAs from Human Serum at JoVE.com

सामन्था Khoury और पामेला Ajuyah ऑस्ट्रेलियाई स्नातकोत्तर पुरस्कार द्वारा समर्थित हैं. हम भी सामन्था Khoury के उनके अतिरिक्त सहायता के लिए translational कैंसर रिसर्च नेटवर्क, Lowy कैंसर रिसर्च सेंटर, न्यू साउथ वेल्स विश्वविद्यालय और उत्तरी translational कैंसर रिसर्च यूनिट को स्वीकार करना होगा.

नोट: स्वस्थ रोगियों या कैंसर के साथ रोगियों से मानव सीरम के नमूने, रॉयल प्रिंस अल्फ्रेड अस्पताल सिडनी (प्रोटोकॉल संख्या X10-0016 और HREC/10/RPAH/24) और प्रौद्योगिकी विश्वविद्यालय से अनुमोदित मानव नैतिक प्रोटोकॉल क?...

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microRNAs की आबादी सीरम में पाया कुल शाही सेना के लगभग 0.4-0.5% का गठन किया. इसके अलावा मानव सीरम में पाया एक उच्च प्रोटीन सामग्री भी है. शाही सेना में सुधार और प्रोटीन और फिनोल दोनों को कम करने के लिए हम कई कदम के सा?...

हाल के वर्षों में, मानव रोगों का जल्दी पता लगाने के लिए उपन्यास बायोमार्कर की खोज के लिए एक से बढ़ धक्का दिया गया है. बहुत ध्यान संभावित मार्कर के रूप में इस तरह के microRNAs 2 (miRNAs या miRs) के रूप में छोटे RNAs का उपयोग पर ध्यान केंद्रित किया गया है. इन छोटे RNAs ऐसे सीरम और अध्ययन के रूप में शरीर के तरल पदार्थ में पाए जाते हैं वे गिरावट को लचीला कर रहे हैं और अलग पर्यावरणीय परिस्थितियों 3 की एक सीमा से अधिक स्थिर रहे हैं पता चला है. इन सुविधाओं, सीरम या परिसंचारी miRNAs आदर्श biomarker 4, 5 को देखते हुए. वर्तमान में जैविक तरल पदार्थ से छोटे आरएनए के अलगाव के लिए दो मुख्य दृष्टिकोण हैं. दूसरा दृष्टिकोण फिनोल और guanidinium thiocyanate अभिकर्मकों 7 के साथ लंबे समय से चली आ रही प्रोटोकॉल का उपयोग करता है, जबकि पहले दृष्टिकोण, छोटे RNAs 6 बाँध और elute करने के लिए स्तंभ आधारित प्रौद्योगिकी का उपयोग करता है. हम मानव सीरम से छोटे RNAs अलग करने के लिए एक सरल, प्रभावी, स्तंभ मुक्त प्रोटोकॉल विकसित किया है. पृथक शाही सेना तुरंत हैडीएनए oligonucleotide सरणियों और शाही सेना अनुक्रमण सहित बहाव के अनुप्रयोगों में ately प्रयोग करने योग्य. सीरम से शाही सेना को अलग करने के लिए फिनोल आधारित विधियों का उपयोग करते समय हम कई मुद्दों के साथ सामना कर रहे थे, क्योंकि यह प्रोटोकॉल विकसित किया गया था. पारंपरिक Chomczynski दृष्टिकोण अक्सर सबसे वाणिज्यिक विक्रेताओं से उपलब्ध अभिकर्मकों की एक सीमा के साथ सबसे प्रयोगशालाओं में प्रयोग किया जाता है. हालांकि उनके व्यापक उपयोग पर विचार, कड़े दिशा निर्देशों लगातार विशेष रक्त या सीरम में, शारीरिक तरल पदार्थों से उच्च गुणवत्ता शाही सेना का उत्पादन करने के लिए विकसित नहीं किया गया है. सीरम से शाही सेना को अलग करने के साथ जुड़े आम समस्याओं कम शाही सेना पैदावार और अलगाव, विशेष रूप से फिनोल के दौरान इस्तेमाल किया अभिकर्मकों के साथ संदूषण शामिल हैं. हमारा दृष्टिकोण इस तरह के मात्रात्मक पीसीआर (qPCR) और शाही सेना अनुक्रमण के रूप में नीचे की ओर विश्लेषण के लिए उच्च गुणवत्ता आरएनए प्रदान करने के लिए इन फिनोल contaminants समाप्त. हम आगे miRNA सरणियों पर इस शाही सेना का परीक्षण किया है.

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			<td height="22" style="height:22px;width:259px;">Name of the Material/Equipment&#160;</td>
			<td style="width:223px;">Company&#160;</td>
			<td style="width:123px;">Catalog Number&#160;</td>
			<td style="width:248px;">Comments/ Description (optional)&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Tri-Reagent RT LS</td>
			<td style="width:223px;">Molecular Research Centre, USA</td>
			<td style="width:123px;">TR 118</td>
			<td style="width:248px;">-</td>
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		<tr height="26">
			<td height="26" style="height:26px;width:259px;">RNase free H20</td>
			<td style="width:223px;">GIBCO Invitrogen</td>
			<td style="width:123px;">10977-023</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Proteinase K&#160;</td>
			<td style="width:223px;">Finnzymes, Finland</td>
			<td style="width:123px;">EO0491</td>
			<td style="width:248px;">-</td>
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			<td height="22" style="height:22px;width:259px;">Heavy Phaselock Tube</td>
			<td style="width:223px;">5PRIME</td>
			<td>2302830</td>
			<td style="width:248px;">2ml capacity</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:259px;">DNA Lobind tube</td>
			<td style="width:223px;">Eppendorf</td>
			<td style="width:123px;">0030 108.078</td>
			<td style="width:248px;">1.5ml capacity</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Glycogen&#160;</td>
			<td style="width:223px;">Invitrogen, USA</td>
			<td style="width:123px;">10814-010</td>
			<td style="width:248px;">5mg/ml</td>
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			<td height="22" style="height:22px;width:259px;">RNA grade Isopropanol&#160;</td>
			<td style="width:223px;">Sigma Aldrich, USA</td>
			<td style="width:123px;">I9516</td>
			<td style="width:248px;">100%</td>
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			<td height="22" style="height:22px;width:259px;">Refrigerated Centrifuge</td>
			<td style="width:223px;">John Morris</td>
			<td style="width:123px;">-</td>
			<td style="width:248px;">-</td>
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			<td height="22" style="height:22px;width:259px;">Nanodrop UV-Vis spectrophotometer</td>
			<td style="width:223px;">Thermo Fisher Scientific, USA</td>
			<td style="width:123px;">-</td>
			<td style="width:248px;">-</td>
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			<td height="22" style="height:22px;width:259px;">RNA grade Ethanol</td>
			<td style="width:223px;">Sigma Aldrich, USA</td>
			<td style="width:123px;">E7023</td>
			<td style="width:248px;">70%</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Agilent 2100 Bioanalyser</td>
			<td style="width:223px;">Agilent, USA</td>
			<td style="width:123px;">-</td>
			<td style="width:248px;">-</td>
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isolation of small noncoding rnas from human serum

शाही सेना और उसकी अभिव्यक्ति का विश्लेषण कई प्रयोगशालाओं में एक आम सुविधा है. महत्व के स्तनधारी कोशिकाओं में पाए जाते हैं जो microRNAs, जैसे छोटे RNAs के उद्भव है. इन छोटे RNAs इस तरह के विकास, विकास और मृत्यु और ज्यादा ब्याज के रूप में महत्वपूर्ण रास्ते को नियंत्रित शक्तिशाली जीन नियामकों शारीरिक तरल पदार्थ में उनकी अभिव्यक्ति के निर्देश पर किया गया है. इस तरह के कैंसर और सीरम बायोमार्कर के रूप में उनके संभावित आवेदन के रूप में मानव रोगों में उनके अनियंत्रण की वजह से है. हालांकि, सीरम में miRNA अभिव्यक्ति की विश्लेषण समस्याग्रस्त हो सकता है. ज्यादातर मामलों में सीरम की मात्रा सीमित है और सीरम छोटे RNAs केवल 0.4-0.5% 1 का गठन जिनमें से कुल शाही सेना के कम मात्रा में होता है. इस प्रकार सीरम से गुणवत्ता शाही सेना की पर्याप्त मात्रा के अलगाव के शोधकर्ताओं के लिए एक बड़ी चुनौती बन गई है. इस तकनीकी पत्र में, हम डीएनए सरणियों या qPCR विश्लेषण के लिए या तो पर्याप्त शाही सेना प्राप्त करने के लिए मानव सीरम की केवल 400 μl का उपयोग करता है जो एक विधि का प्रदर्शन. एकइस विधि के dvantages अपनी सादगी और उच्च गुणवत्ता शाही सेना उपज करने की क्षमता है. यह छोटे RNAs की शुद्धि के लिए कोई विशेष स्तंभों की आवश्यकता है और आम प्रयोगशालाओं में पाया जनरल अभिकर्मकों और हार्डवेयर का इस्तेमाल करता. हमारे विधि एक ही समय में उच्च गुणवत्ता शाही सेना से बेदखल करते हुए फिनोल प्रदूषण को खत्म करने के लिए एक चरण ताला जेल का इस्तेमाल करता. हम भी आगे अलगाव चरण के दौरान सभी को दूर करने के लिए एक अतिरिक्त कदम परिचय. इस प्रोटोकॉल सीरम से 100 एनजी / μl की कुल शाही सेना की पैदावार को अलग करने में बहुत प्रभावी है, लेकिन यह भी अन्य जैविक ऊतकों के लिए अनुकूलित किया जा सकता.

चित्रा 1 सीरम से अलग शाही सेना के एक ठेठ यूवी / विज़ स्पेक्ट्रम का प्रतिनिधित्व करता है. इस प्रोफाइल से हम क्रमशः, दोनों 270 एनएम और 230 एनएम पर फिनोल और जैविक contaminants के साथ 280 एनएम प्रोटीन संदूषण का उल्लेख ...

Watch this Scientific Journal Video about Isolation of Small Noncoding RNAs from Human Serum at JoVE.com

Isolation of Small Noncoding RNAs from Human Serum

इस प्रोटोकॉल मानव सीरम से छोटे RNAs निकालने के लिए एक विधि का वर्णन करता है. हम डीएनए सरणियों में इस्तेमाल के लिए कैंसर सीरम से microRNAs अलग और भी singleplex मात्रात्मक पीसीआर के लिए इस विधि का इस्तेमाल किया है. प्रोटोकॉल उच्च गुणवत्ता शाही सेना उपज के लिए संशोधनों के साथ फिनोल और guanidinium thiocyanate अभिकर्मकों का इस्तेमाल करता.

मानव सीरम से लघु noncoding RNAs के अलगाव

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

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Research

JoVE Journal

Biology

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

मानव शवपरीक्षा मस्तिष्क के ऊतकों से neuronal नाभिक अलगाव

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

मानव अग्नाशय के कैंसर xenografts से स्टेम सेल के अलगाव

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

और वयस्क मानव त्वचा से उपकला स्टेम सेल के अलगाव संस्कृति

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

लघु अणु प्रेरण के साथ pluripotent मानव भ्रूण स्टेम कोशिकाओं से मानव कार्डिएक व्यापारियों और cardiomyocytes के कुशल व्युत्पत्ति

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

मानव Lipoaspirates से वसा व्युत्पन्न स्टेम कोशिकाओं के मैनुअल अलगाव

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

माउस और मानव आंतों के ऊतकों से प्राथमिक myofibroblasts का अलगाव

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

माउस और मानव घुटकी से myofibroblasts का अलगाव

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

वाइब्रेटोम-कट मायोकार्डियल स्लाइस से ह्यूमन वेंट्रिकुलर कार्डियोमायोसाइट्स का अलगाव

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Isolation of Human Neutrophils from Whole Blood and Buffy Coats

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells recruited at the site of inflammation via rapid extravasation through vessels. There, neutrophils perform an array of functions to kill invading pathogens and mediate immune signaling. Freshly purified neutrophils from human blood are the model of choice for study, as no cell line fully replicates PMN functions and biology. However, neutrophils are short-lived, terminally differentiated cells and are highly susceptible to activation in response to physical (temperature, centrifugation speed) and biological (endotoxin, chemo- and cytokines) stimuli. Therefore, it is crucial to follow a standardized, reliable, and fast method to obtain pure and non-activated cells. This protocol presents an updated protocol combining density gradient centrifugation, red blood cell (RBC) sedimentation, and RBC lysis to obtain high PMN purity and minimize cell activation. Furthermore, methods to assess neutrophil isolation quality, viability, and purity are also discussed.

This protocol details a method for the isolation of neutrophils from whole blood, buffy coats, or leukapheresis membranes, achieving good yield, high purity, and minimal cell activation. We utilize gradient purification, red blood cell (RBC) sedimentation, and RBC lysis to obtain a high-quality/purity neutrophil preparation.

पूरे रक्त और बफी कोट से मानव न्यूट्रोफिल का अलगाव

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells ...

Isolation of Epithelial Cells from Human Dental Follicle

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while &#945;-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells.
The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

The dental follicle contains an epithelial population and mesenchymal cells. The epithelial population was selected from the heterogeneous dental follicle cell population by providing a distinct culture medium. Epithelial cells survived and formed colonies in a serum-free medium.

मानव दंत कूप से उपकला कोशिकाओं का अलगाव

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was ...