Name: expansion and adipogenesis induction of adipocyte progenitors from perivascular adipose tissue isolated by magnetic activated cell sorting
Uploaded: 2017-06-30T14:00:00
Description: Watch this Scientific Journal Video about Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting at JoVE.com

Contreras og Watts Laboratories og Dr. William Raphael. Disse forsøkene ble støttet av NHLBI F31 HL128035-01 (vevsfordøyelsesprotokoll standardisering), NHLBI 5R01HL117847-02 og 2P01HL070687-11A1 (dyr) og NHLBI 5R01HL117847-02 (celleisolasjon og kultur).

Alle prosedyrer beskrevet i dette dokumentet følger retningslinjer fastsatt av Institutt for dyrepleie og brukskomité (IACUC) fra Michigan State University. Alle buffere og medier bør beskyttes mot lys. 1. Fremstilling av buffere, medier og instrumenter <ol><li> Klargjør Krebs Ringer Bikarbonatbuffert løsning (KRBB): 135 mM natriumklorid, 5 mM kaliumklorid, 1 mM magnesiumsulfat, 0,4 mM kaliumfosfatdibasisk, 5,5 mM glukose, 1% antibiotisk / antimykotisk (10 000 enheter / ml penicillin,...

<ol>
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Det sentrale fokuset i foreliggende eksperiment er isolering, ekspansjon og adipogen induksjon av APC fra PVAT-depoter. Her presenterer vi en protokoll for isolering av APC basert på identifisering av celler som uttrykker overflate markørene CD34 og PDGFRα. Disse overflateproteiner ble tidligere identifisert på APC med høye proliferasjonshastigheter og potensialet til å differensiere til hvite eller brune adipocytter i forskjellige adipose depots 14 , 15 ....

Perivaskulær Adipose Tissue (PVAT), på grunn av sin nærhet til blodkar, er en viktig parakrin signalering komponent i vaskulatur funksjon 1 . Utvidelse av dette fettvev er avhengig av fenotypen av Adipocyte Progenitor Cells (APC) tilstede 2 , 3 . Isolering av celler fra fettvev er vanskelig da primære adipocytter er skjøre, flytende og varierer i størrelse. Visse isolasjonsteknikker kan også endre cellefenotype og morfologi ved å øke inflammatorisk proteinsyntese og redusere adipogen genuttrykk 4 , understreker betydningen av en protokoll som opprettholder integriteten til cellene. Kultur av primære celler og spesifikke preadipocyt-subpopulasjoner gir en reduktivistisk tilnærming til vekst i in vivo og opprettholder ekvivalent cellulær genetisk sminke 5 , selv om man arbeider tiMeg med disse cellene er begrenset på grunn av forverring med aldring eller senescence 6 . Preadipocytter fra forskjellige adipose depots, inkludert subkutane og omental depoter, viser også forskjeller i proliferation 7 , som understreker viktigheten av å samle celler fra bestemte anatomiske steder. Forløperceller fra ikke-PVAT hvite adipose depoter har blitt preget av tidligere studier 7 , 8 , 9 , men mindre er kjent om PVAT APC fenotyper. Teknologiene beskrevet her tillater analyse av spesifikke og definerte APC-populasjoner med minimal innvirkning på deres morfologi, levedyktighet og potensial til å proliferere og differensiere. Magnetisk aktivert cellesortering (MCS) er egnet til nedstrømsapplikasjoner, for eksempel kultur, da perlene oppløses uten å endre cellen. MCS er også økonomisk, og når antistoffet conSentreringer har blitt standardisert, behovet for strømningscytometrianalyser er minimal. In vitro- studier med PVAT-forløpere kan også gi et glimt av potensialet som disse primære cellene kan ha.

<table border="0" cellpadding="0" cellspacing="0" width="757">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:325px;">Tissue Dissection</td>
			<td style="width:168px;"></td>
			<td style="width:108px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting Dishes</td>
			<td>Handmade with Silicone</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Culture Petri Dish</td>
			<td>Pyrex</td>
			<td></td>
			<td>7740 Glass</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silicone Elastomer</td>
			<td>Dow Corning</td>
			<td>Sylgard 170</td>
			<td>Kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Braided Silk Suture</td>
			<td>Harvard Apparatus</td>
			<td>51-7615</td>
			<td>SP104</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereomicroscope MZ6</td>
			<td>Leica</td>
			<td></td>
			<td>10447254</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereomaster Microscope Fiber-Optic Light Source</td>
			<td>Fisher Scientific</td>
			<td>12562-36</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vannas Scissors</td>
			<td>George Tiemann &#38; Co</td>
			<td>160-150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Splinter Forceps</td>
			<td>George Tiemann &#38; Co</td>
			<td>160-55</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Scissors</td>
			<td>George Tiemann &#38; Co</td>
			<td>105-400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KRBB Solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>7487-88-9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium Phosphate Dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>7758-114</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>50-99-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic/Antimicotic</td>
			<td>Corning</td>
			<td>30-004-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Corning</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Digestion</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase Type 1</td>
			<td>Worthington Biochemical</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin</td>
			<td>Fisher Scientific</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Red Blood Cell Lysis Buffer</td>
			<td>BioLegend</td>
			<td>420301</td>
			<td>1X Working Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water Bath</td>
			<td>Thermo-Fisher Scientific</td>
			<td>2876</td>
			<td>Reciprocal Shaking Bath</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biosafety Cabinet</td>
			<td>Thermo-Fisher Scientific</td>
			<td>1385</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rotisserie Incubator</td>
			<td>Daigger</td>
			<td>EF4894C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 &#181;m Cell Strainer</td>
			<td>Thermo-Fisher Scientific</td>
			<td>22-363-549</td>
			<td>Yellow</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40 &#181;m Cell Strainer</td>
			<td>Thermo-Fisher Scientific</td>
			<td>22-363-547</td>
			<td>Blue</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hemocytometer</td>
			<td>Cole-Parmer</td>
			<td>UX-79001-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypan Blue</td>
			<td>Sigma-Aldrich</td>
			<td>93595</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Isolation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OctoMACS Kit</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-108</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">(DMEM):F12 Medium</td>
			<td>Corning</td>
			<td>90-090</td>
			<td>Medium Base</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35016CV</td>
			<td>USA Origins</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal Donkey Serum</td>
			<td>AbCam</td>
			<td>AB7475</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CD34</td>
			<td>Santa Cruz</td>
			<td>SC-7324</td>
			<td>FITC conjugated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-PDGFR&#945;</td>
			<td>Thermo-Fisher Scientific</td>
			<td>PA5-17623</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey Anti-Rabbit IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>712-007-003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS 10X</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td>1X Working Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Fisher Scientific</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CO2 Cell Incubator</td>
			<td>Thermo-Fisher Scientific</td>
			<td>51030285</td>
			<td>Heracell 160i&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-Well Plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">48- Well Plates</td>
			<td>Corning</td>
			<td>3548</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-Well Plates, Black Wall</td>
			<td>Corning</td>
			<td>353376</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>144-55-8</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Calf Serum</td>
			<td>Corning</td>
			<td>35011CV</td>
			<td>USA Origins</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>50-81-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>58-85-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pantothenate</td>
			<td>Sigma-Aldrich</td>
			<td>137-08-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Corning</td>
			<td>61-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bone Morphogenic Protein 4</td>
			<td>Prospec Bio</td>
			<td>CYT-081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epidermal Growth Factor</td>
			<td>PeproTech</td>
			<td>400-25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leukemia Inhibitory Factor</td>
			<td>PeproTech</td>
			<td>250-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platelet-derived Growth Factor BB</td>
			<td>Prospec Bio</td>
			<td>CYT-740</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Basic Fibroblast Growth Factor</td>
			<td>PeproTech</td>
			<td>450-33</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin</td>
			<td>Corning</td>
			<td>25-800-CR</td>
			<td>ITS Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IBMX</td>
			<td>Sigma-Aldrich</td>
			<td>28822-58-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>50-02-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T3 (Triiodothyronine)</td>
			<td>Sigma-Aldrich</td>
			<td>6893-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CyQUANT Proliferation Assay</td>
			<td>Thermo-Fisher Scientific</td>
			<td>C7026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AdipoRed Fluorescence Assay Reagent</td>
			<td>Lonza</td>
			<td>PT-7009</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oil Red O Lipid Dye Reagent</td>
			<td>Sigma-Aldrich</td>
			<td>O1391</td>
			<td>In Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M1000 Microplate Reader</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eclipse Inverted Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digital Sight DS-Qil Camera</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

expansion and adipogenesis induction of adipocyte progenitors from perivascular adipose tissue isolated by magnetic activated cell sorting

Utvidelse av perivaskulær adiposvæv (PVAT), en stor regulator av vaskulær funksjon gjennom parakrinsignalering, er direkte relatert til utvikling av hypertensjon under fedme. Omfanget av hypertrofi og hyperplasi er avhengig av depotplassering, kjønn, og typen av adipocytprogenitorcelle (APC) fenotyper tilstede. Teknikker brukt til APC og preadipocytter isolasjon de siste 10 årene har drastisk forbedret nøyaktigheten ved hvilke enkelte celler kan identifiseres basert på spesifikke celleoverflate markører. Imidlertid kan isolering av APC og adipocytter være en utfordring på grunn av cellens skjøthet, spesielt hvis den intakte celle beholdes for cellekulturapplikasjoner. Magnetisk aktivert cellesortering ( MCS) gir en metode for å isolere større antall levedyktig APC per vektenhet av fettvev. APC høstet av MCS kan brukes til in vitro- protokoller for å utvide preaDipocytter og differensiere dem til adipocytter ved bruk av vekstfaktor-cocktailer som muliggjør analyse av det potensielle og adipogene potensialet som beholdes av cellene. Dette eksperimentet fokuserte på aorta- og mesenteriske PVAT-depotene, som spiller sentrale roller i utviklingen av kardiovaskulær sykdom under ekspansjon. Disse protokollene beskriver metoder for å isolere, utvide og differensiere en definert befolkning av APC. Denne MCS-protokollen tillater isolasjon å brukes i ethvert eksperiment hvor celle sortering er nødvendig med minimal utstyr eller trening. Disse teknikkene kan bistå ytterligere eksperimenter for å bestemme funksjonaliteten til spesifikke cellepopulasjoner basert på tilstedeværelsen av celleoverflate markører.

Proliferativ kapasitet av preadipocytter og adipogen potensial for adipocytprekursorer er karakteristika som opprettholdes in vitro 11 . In vitro- proliferasjon av isolert SVF og APC fra aPVAT, mPVAT og GON av hannrotter ble evaluert ved 8, 24, 48 og 96 timer etter plating ved bruk av et kvantitativt DNA-assay. Ingen stedforskjeller i SVF-ekspansjonshastigheten ble observert når som helst, unntatt APC fra aPVAT, som hadde mindre proliferasjon p?...

Watch this Scientific Journal Video about Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting at JoVE.com

Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

Her rapporterer vi en metode for isolering av Adipocyte Progenitor Cell (APC) populasjoner fra Perivascular Adipose Tissue (PVAT) ved hjelp av magnetisk aktivert cellesortering (MCS). Denne metoden tillater økt isolering av APC per gram fettvev i sammenligning med fluorescensaktivert cellesortering (FACS).

Ekspansjon og adipogeneseinduksjon av adipocytprogenitorer fra perivaskulær adiposevev isolert ved magnetisk aktivert cellesortering

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the ...

Research

JoVE Journal

Developmental Biology

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Isolasjon og Differensiering av adipose-avledet stamceller fra svine Subkutan fettvev

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Isolasjon og utvidelse av Mesenchymale Stem / stromal celler avledet fra human placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Isolering av perivaskulær multipotente forløpercelle populasjoner fra humane kardiale Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Induksjon av akutt Skeletal Muscle Regenerering av Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Chondrogenic Differensiering Induksjon av adipose-avledet stamceller ved Sentrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Identifikasjon av erytromyeloidprogenitorer og deres avkom i musembryoen ved flowcytometri

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

Hele-celle Patch-clamp Opptak av isolerte primære epitelceller fra epididymene

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generasjon av stillaset-fri, tredimensjonale Insulin uttrykke Pancreatoids fra musen bukspyttkjertelen Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Effektiv generasjon av bukspyttkjertelen/tolvfingertarmen Homeobox Protein 1+ bakre Foregut/Pancreatic Progenitors fra hPSCs i vedheft kulturer

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

Isolasjon, kultur og adipogen induksjon av Neural Crest Original Adipose-Avledet stamceller fra periaortisk fettvev

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...