Name: preparation of adipose progenitor cells from mouse epididymal adipose tissues
Uploaded: 2020-08-25T14:45:00
Description: Watch this Scientific Journal Video about Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues at JoVE.com

Vi anerkjenner Mayo Clinic Microscopy Cell Analysis Core Flow Cytometry Facility for hjelp med FACS sortering.

Alle dyreforsøksprosedyrer ble utført under godkjenning av Mayo Clinic Institutional Animal Care and Use Committee.
1. Forberedelse av oppløsning 
<ol><li>Forbered kollagen 2 % (w/v) oppløsning ved å oppløse kollagenase II 2 g i 100 ml Hanks balanserte saltoppløsning (HBSS). Aliquot 200 μL hver for hver bruk.</li>
	<li>Forbered nøytraliseringsmediet ved å blande 84 ml F-12 medium, 15 ml hesteserum og 1 ml penicillin/streptomycin.</li>
	<li>Forbered strømningscytom...

<ol>
	<li>Krotkiewski, M., Bjorntorp, P., Sjostrom, L., Smith, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+obesity+on+metabolism+in+men+and+women.+Importance+of+regional+adipose+tissue+distribution.">Impact of obesity on metabolism in men and women. Importance of regional adipose tissue distribution.</a> The Journal of Clinical Investigation. 72, 1150-1162 (1983).</li><li>Salans, L. B., Horton, E. S., Sims, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+obesity+in+man:+cellular+character+of+the+adipose+tissue.">Experimental obesity in man: cellular character of the adipose tissue.</a> The Journal of Clinical Investigation. 50, 1005-1011 (1971).</li><li>Trayhurn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+adipose+tissue+function+and+dysfunction+in+obesity.">Hypoxia and adipose tissue function and dysfunction in obesity.</a> Physiological Reviews. 93, 1-21 (2013).</li><li>Halberg, N., 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=Hypoxia-inducible+factor+1alpha+induces+fibrosis+and+insulin+resistance+in+white+adipose+tissue.">Hypoxia-inducible factor 1alpha induces fibrosis and insulin resistance in white adipose tissue.</a> Molecular and Cellular Biology. 29, 4467-4483 (2009).</li><li>Upadhyay, J., Farr, O., Perakakis, N., Ghaly, W., Mantzoros, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+as+a+Disease.">Obesity as a Disease.</a> Medical Clinics of North America. 102, 13-33 (2018).</li><li>Cawthorn, W. P., Scheller, E. L., MacDougald, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+stem+cells+meet+preadipocyte+commitment:+going+back+to+the+future.">Adipose tissue stem cells meet preadipocyte commitment: going back to the future.</a> Journal of Lipid Research. 53, 227-246 (2012).</li><li>Cho, D. S., Lee, B., Doles, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refining+the+adipose+progenitor+cell+landscape+in+healthy+and+obese+visceral+adipose+tissue+using+single-cell+gene+expression+profiling.">Refining the adipose progenitor cell landscape in healthy and obese visceral adipose tissue using single-cell gene expression profiling.</a> Life Science Alliance. 2, 201900561 (2019).</li><li>Burl, R. B., 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=Deconstructing+Adipogenesis+Induced+by+beta3-Adrenergic+Receptor+Activation+with+Single-Cell+Expression+Profiling.">Deconstructing Adipogenesis Induced by beta3-Adrenergic Receptor Activation with Single-Cell Expression Profiling.</a> Cell Metabolism. 28, 300-309 (2018).</li><li>Hepler, C., 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+functionally+distinct+fibro-inflammatory+and+adipogenic+stromal+subpopulations+in+visceral+adipose+tissue+of+adult+mice.">Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice.</a> eLife. 7, 39636 (2018).</li><li>Merrick, D., 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+a+mesenchymal+progenitor+cell+hierarchy+in+adipose+tissue.">Identification of a mesenchymal progenitor cell hierarchy in adipose tissue.</a> Science. 364, 2501 (2019).</li><li>Schwalie, P. C., 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=A+stromal+cell+population+that+inhibits+adipogenesis+in+mammalian+fat+depots.">A stromal cell population that inhibits adipogenesis in mammalian fat depots.</a> Nature. 559, 103-108 (2018).</li><li>Raajendiran, A., 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+Metabolically+Distinct+Adipocyte+Progenitor+Cells+in+Human+Adipose+Tissues.">Identification of Metabolically Distinct Adipocyte Progenitor Cells in Human Adipose Tissues.</a> Cell Reports. 27, 1528-1540 (2019).</li><li>De Matteis, R., 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=In+vivo+physiological+transdifferentiation+of+adult+adipose+cells.">In vivo physiological transdifferentiation of adult adipose cells.</a> Stem Cells. 27, 2761-2768 (2009).</li><li>Hanamsagar, R., 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=An+optimized+workflow+for+single-cell+transcriptomics+and+repertoire+profiling+of+purified+lymphocytes+from+clinical+samples.">An optimized workflow for single-cell transcriptomics and repertoire profiling of purified lymphocytes from clinical samples.</a> Scientific Reports. 10, 2219 (2020).</li><li>Marinovic, M. P., 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=Crotamine+induces+browning+of+adipose+tissue+and+increases+energy+expenditure+in+mice.">Crotamine induces browning of adipose tissue and increases energy expenditure in mice.</a> Scientific Reports. 8, 5057 (2018).</li><li>Takahashi, H., 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=Biological+and+clinical+availability+of+adipose-derived+stem+cells+for+pelvic+dead+space+repair.">Biological and clinical availability of adipose-derived stem cells for pelvic dead space repair.</a> Stem Cells Translational Medicine. 1, 803-810 (2012).</li><li>Cowan, C. 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=Adipose-derived+adult+stromal+cells+heal+critical-size+mouse+calvarial+defects.">Adipose-derived adult stromal cells heal critical-size mouse calvarial defects.</a> Nature Biotechnology. 22 (5), 560-567 (2004).</li><li>Hagberg, C. E., 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=Flow+Cytometry+of+Mouse+and+Human+Adipocytes+for+the+Analysis+of+Browning+and+Cellular+Heterogeneity.">Flow Cytometry of Mouse and Human Adipocytes for the Analysis of Browning and Cellular Heterogeneity.</a> Cell Reports. 24, 2746-2756 (2018).</li></ol>

Enkeltcellede RNA-sekvensering (scRNA-seq) får raskt trekkraft som et kraftig verktøy for samtidig å studere ulike cellepopulasjoner på enkeltcellenivå. På grunn av høye kostnader forbundet med prøveforberedelse og høy gjennomstrømningssekvensering, er det viktig å optimalisere cellulære innganger (høy levedyktighet og renhet) for å øke sannsynligheten for eksperimentell suksess. Noen celleforberedelsesprotokoller er avhengige av fjerning av døde celler og rusk ved hjelp av lavspinnvasker og kolonnebasert...

Fettvev spiller en nøkkelrolle i energimetabolismen. Overflødig energi lagres i form av lipider, og fettvev er i stand til betydelig ekspansjon eller tilbaketrekking avhengig av ernæringsstatus og energisk etterspørsel. Utvidelse av fettvev kan skyldes en økning i adipocyttstørrelse (hypertrofi) og/eller fra en økning i adipocyttnummer (hyperplasi); den sistnevnte prosessen tett regulert av spredning og differensiering av fett stamceller1,2. Under fedme, fettvev utvider seg overdrevet, og vev dysfunksjon - inkludert hypoksi, betennelse, og insulinresistens – utvikler ofte3,4. Disse komplikasjonene er risikofaktorer for mange kroniske sykdommer, inkludert hypertensjon, diabetes, kardiovaskulære sykdommer, hjerneslag og kreft5. Derfor er det å begrense ukontrollert fettvevsutvidelse og redusere fettvevspatologier topp biomedisinske forskningsprioriteringer. Under fettvevsutvidelse sprer resident fettvevsavledede stamceller (ASCer) seg og differensierer sekvensielt til preadipocytter (engasjerte stamceller) og deretter til modne adipocytter6. Nyere encellede RNA-sekvenseringsstudier (scRNA-seq) viser at disse fettstamtamcellepopulasjonene (APC) (ASC og preadipocytter) viser betydelig molekylær og funksjonell heterogenitet7,8,9,10,11,12. AsCs viser for eksempel en redusert adipogenic differensieringskapasitet, samtidig som den viser høyere sprednings- og ekspansjonsfunksjoner, sammenlignet med preadipocytter7. Ytterligere molekylære forskjeller rapporteres innenfor ASC- og preadipocyttpopulasjoner, selv om den funksjonelle relevansen av disse forskjellene fortsatt er uklar7. Sammen fremhever disse dataene kompleksiteten i fettstamcelleutvalget og understreker behovet for å utvikle og standardisere verktøy for å bedre forstå og manipulere disse kritiske cellepopulasjonene.
Denne protokollen beskriver isoleringen av høy levedyktighet Sca1+ fett stamcellepopulasjoner fra mus epididymal fett pads som er egnet for sensitive nedstrøms analyser, inkludert encellede studier (scRNA-sekvensering) og cellekultur. Isolasjon og dissosiasjon av epididymale fettputer ble utført som tidligere beskrevet7,13 med små modifikasjoner som forbedrer levedyktigheten til isolerte APCer. Kort sagt er dissosierte celler fra epididymale fettputer farget med antistoffer mot Sca1, en markør for både ASC og preadipocytter6,7og andre lineage (Lin) markører: Ter119 (erythroid celler), CD31 (endotelceller) og CD45 (leukocytter). Levedyktig Sca1+/Ter119-/CD31-/CD45-/DAPI- celler sorteres deretter etter fluorescensaktivert cellesortering (FACS). Viktigere, denne protokollen ble validert ved vellykket isolasjon og analyse av levedyktig Sca1+/ Lin- fett stamceller rapportert i en nylig enkeltcellede RNA sekvensering studie som identifiserte funksjonelt heterogene underpopulasjoner innenfor ASCs og preadipocytes7.

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	<tbody>
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			<td height="21" style="height:21px;width:332px;">1.7 mL microcentrifuge tube</td>
			<td style="width:181px;">VWR</td>
			<td style="width:119px;">87003-294</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">13 mL culture tube</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">50-809-216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">15 mL conical tube</td>
			<td style="width:181px;">Greiner Bio-one</td>
			<td style="width:119px;">188 271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">5 mL test tube with cell strainer snap cap</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">08-771-23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">50 mL conical tube</td>
			<td style="width:181px;">Greiner Bio-one</td>
			<td style="width:119px;">227 261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">70 &#181;m cell strainer</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">22-363-548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-CD31-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-519</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-CD45-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-491</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-Sca1-APC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-Ter119-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-112-908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">BSA</td>
			<td style="width:181px;">Gold Biotechnology</td>
			<td style="width:119px;">A-420-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Collagenase type II</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">17101-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">DAPI</td>
			<td style="width:181px;">Thermo Fisher</td>
			<td style="width:119px;">D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">F-12 medium</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11765-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">FcR blocking reagent</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-092-575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Hanks&#39; balanced salt solution (HBSS)</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14025-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Horse serum</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">16050-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Penicillin-streptomycin</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Propidium iodide solution</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-233</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of adipose progenitor cells from mouse epididymal adipose tissues

Fedme og metabolske forstyrrelser som diabetes, hjertesykdom og kreft, er alle forbundet med dramatisk fettvev remodeling. Vev-resident fett stamceller (APCer) spille en nøkkelrolle i fettvev homeostase og kan bidra til vev patologi. Den økende bruken av encellede analyseteknologier – inkludert encellede RNA-sekvensering og encellede proteomikk – forvandler stamcellefeltet ved å tillate enestående oppløsning av individuelle celleuttrykksendringer i sammenheng med endringer i hele befolkningen eller vev. I denne artikkelen gir vi detaljerte protokoller for å dissekere mus epididymal fettvev, isolere enkelt fettvev-avledede celler, og utføre fluorescens aktivert celle sortering (FACS) for å berike for levedyktig Sca1+/ CD31-/ CD45-/ Ter119- APCer. Disse protokollene vil tillate etterforskere å forberede høy kvalitet APCer egnet for nedstrøms analyser som enkeltcellede RNA sekvensering.

Fire måneder gamle hann FVB-mus ble brukt i dette eksperimentet. Etter utelukkelse av rusk og doble ved hjelp av FSC/ SSC tomter, levedyktige celler (DAPI- befolkning) ble inngjerdet, etterfulgt av valg av APC+/ FITC- populasjon (figur 1). DAPI-, APC- og FITC-portene ble trukket basert på den uopphetede kontrollen. Gating strategier er vist i figur 1.
Etter 1 t sortering ble isolasjonskvaliteten kvan...

Watch this Scientific Journal Video about Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues at JoVE.com

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Vi presenterer en enkel metode for å isolere svært levedyktige fett stamceller fra mus epididymal fett pads ved hjelp av fluorescens aktivert celle sortering.

Fremstilling av fett stamceller fra mus epididymal fettvev

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

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Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

Manuell Isolering av adipose-avledet stamceller fra menneske Lipoaspirates

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

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Differensiering kapasitet på menneskelig aorta Perivascular liggende under Adipose Progenitor celler

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

Identification and Dissection of Diverse Mouse Adipose Depots

Identifisering og Disseksjon av diverse mus fettlagre

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Vurdering av vaskulær tone respons ved hjelp av isolerte Chrezbryzheechno arterier med fokus på modulering av inflammasjon fettvev

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Isolering, proliferasjon og differensiering av Rhesus Macaque fettavledede stamceller

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Isolering og identifisering av vaskulære endotelceller fra distinkte fettdepoter for nedstrømsapplikasjoner

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

Adipose Tissue-Derived Stem Cell Isolation and Characterization

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

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Isolering og identifisering av mesenkymale stamceller avledet fra fettvev av Sprague Dawley-rotter

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...