Name: digestion of whole mouse eyes for multi-parameter flow cytometric analysis of mononuclear phagocytes
Uploaded: 2020-06-17T14:45:00
Description: Watch this Scientific Journal Video about Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes at JoVE.com

JAL blev støttet af NIH tilskud K08EY030923; CMC blev støttet af et K01-tilskud (5K01AR060169) fra NIH National Institute of Arthritis and Musculoskeletal Diseases og en Novel Research Grant (637405) fra Lupus Research Alliance.

Alle procedurer blev godkendt af Northwestern University Institutional Animal Care and Use Committee. C57BL/6 mus var gruppehus på en barriere facilitet på Center for Comparative Medicine på Northwestern University (Chicago, IL). Alle dyr blev opstaldet i 12/12 timers lys / mørk cyklus med fri adgang til mad og vand.
1. Indsamling af okulært væv
<ol><li>Ofre 10-12 uger gamle C57BL/6J mus af begge køn ved hjælp af reguleret administration af kuldioxid, indtil musen ikke reagerer og ik...

<ol>
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Multiparameterflowcytometri giver mulighed for kvantitativ analyse af flere celletyper i et komplekst væv. I denne rapport beskriver vi flowcytometrisk detektion af mononukleare fagocytpopulationer, herunder monocytter, dendritiske celler og makrofager, efter laserskade i museøjet. Liyanage et al nylig rapporteret en lignende gating strategi til påvisning af neutrofiler, eosinofiler, lymfocytter, DC'er, makrofager, infiltrere makrofager, og monocytter27. Vores gating strategi primært adskiller...

Det medfødte immunsystem omfatter flere celletyper, der stimulerer komplementaktivering og inflammation. Medfødte immunceller omfatter naturlige dræberceller (NK), mastceller, basofiler, eosinofiler, neutrofiler og mononukleare fagocytter. Mononukleare fagocytter, som består af monocytter, makrofager og dendritiske celler, har været impliceret i patofysiologien af flere oftalmiske tilstande, herunder uveitis, diabetisk retinopati og aldersrelateret makuladegeneration (AMD)1. I denne protokol vil vi fokusere på identifikation af mononukleare fagocytter ved hjælp af multiparameterflow cytometrisk analyse i en musemodel af neovaskulær AMD2. Denne protokol kan tilpasses til musemodeller af diabetisk retinopati og / eller uveitis, men mere omfattende okulær dissektion anbefales på grund af disse sygdommes systemiske karakter.
Mononukleare fagocytter udtrykker overlappende celleoverflademarkører. Langvarige makrofager i væv og mikroglia stammer fra den erythromyeloide stamfader, der stammer fra æggeblommen3, mens genanvendelse af makrofager og dendritiske celler adskiller sig fra den knoglemarvsafledte makrofaglige cellegenitor4. Musecelleoverflademærker, der er fælles for monocytter, makrofager og dendritiske celler, omfatter CD45, CD11b5, F4/806, Cx3cr17og den intracellulære markør Iba18. For at overvinde denne udfordring definerer transskriptionsanalyse af makrofager, monocytter og dendritiske celler fra flere væv CD64 som en makrofaglig celleoverflademarkør6. Makrofager er blevet beskrevet i iris, choroid, ciliary krop, og synsnerven i sunde øjne3. Alternativt er identifikation af dendritiske celler vanskeligere. den mest specifikke metode til identifikation af dendritiske celler kræver skæbnekortlægning ved hjælp af Zbtb46-GFP-reportermusen9. Uafhængigt af denne reporterlinje kan udtryk for CD11c og MHCII i forbindelse med fraværet af CD64 identificerepotentielledendritiske celler 6,10. Dendritiske celler er blevet identificeret i hornhinden, bindehinden, iris, og choroid i normale øjne11. Microglia er specialiserede makrofager placeret i nethinden, beskyttet af blod-nethinde barrieren, og stammer fra æggeblomme sæk stamceller12. Som følge heraf kan nethindemikroglia skelnes fra monocyt-afledte makrofager ved deres dimniveauer af CD45-udtryk13 og høje niveauer af Tmem119, som fås som et flowcytometriantistof antistof14. Ved microglia aktivering, dog CD45 kan up-reguleret15 og Tmem119 kan være ned-reguleret3, der viser kompleksiteten af microglia biologi, og dette er sandsynligvis relevant i både AMD og dens musemodel. Endelig kan monocytter opdeles i mindst to undertyper, herunder klassisk og ikke-klassisk. Klassiske monocytter viser CCR2+Ly6ChøjCX3CR1lavt udtryk, og ikke-klassiske monocytter demonstrere CCR2-Ly6ClavCX3CR1høje markører5.
På grund af nødvendigheden af den kvantitative analyse af markørudtryk, dvs. Yderligere fordele omfatter identifikation af underpopulationer, evnen til at bruge fluorescensaktiveret cellesortering (FACS) til at sortere cellepopulationer til transskriptionomic eller proteomic analyse og skæbnekortlægning. Den største ulempe ved multi-parameter flow cytometry er manglen på vævsarkitektur. Dette kan overvindes ved oftalmisk dissektion i de forskellige okulære subcompartments: hornhinde, bindehinde, iris, linse, nethinden, og choroid-sclera kompleks. Derudover kan bekræftende immunfluorescens imaging udføres, men er begrænset af antallet af markører og mangel på robust kvantantitation.
Genom brede forening undersøgelser har knyttet flere komplement gener med AMD16. Komplement aktivering fører til anafylakin produktion, leukocyt rekruttering, og deraf følgende inflammation. I komplementreceptor mangelfulde mus reducerer laserskade mononuklear phagocytrekruttering og laserinduceret choroidal neovascularisering (CNV) område17. Tilsvarende C-C motiv kemokin receptor 2 (CCR2) knockout mus, som er mangelfuld i monocyt rekruttering til vævet, viser både nedsat mononuklear phagocyt rekruttering og laser-induceret CNV område18. Disse data link supplement og mononukleare fagocytter med eksperimentelLE CNV og muligvis neovaskulær AMD. Til støtte for denne forening dysreguleres komplementreceptorer på perifere blodmon monocytter hos patienter med neovaskulær AMD19,20. Disse data viser en stærk sammenhæng mellem AMD og mononukleare fagocytter.
I dette manuskript vil vi bruge den eksperimentelle laserinducerede CNV-model til at karakterisere de mononukleare fagocytpopulationer i museøjet ved hjælp af multiparameterflowcytometri. Laser-induceret CNV er standardmus model af neovaskulær AMD, som viste effekten af den nuværende første linje neovaskulær AMD behandling21. Denne protokol vil beskrive enucleation af museøjne, okulær dissektion, fordøjelse i en enkelt celle suspension, antistof farvning, bestemmelse af laser spændinger ved hjælp af enkelt farve kontrol, og gating strategi ved hjælp af fluorescens minus en (FMO) kontrol. En detaljeret beskrivelse af den laserfremkaldte CNV-model findes i en tidligere publikation22. Ved hjælp af denne protokol definerer vi mikroglia, monocytter, dendritiske celler og makrofagpopulationer. Desuden vil vi bruge MHCII og CD11c til yderligere at definere makrofag delsæt inden for laser induceret CNV model.

<table>
	<tbody>
		<tr>
			<td>0.6 ml microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-120</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 ml microcentrifuge tubes</td>
			<td>Costar</td>
			<td>3620</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml pipettes</td>
			<td>Fisher Scientific</td>
			<td>431031</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conicals</td>
			<td>ThermoFisher</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>1x HBSS, 500 ml</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5 ml syringe</td>
			<td>Henke Sass Wolf</td>
			<td>7886-1</td>
			<td></td>
		</tr>
		<tr>
			<td>20% paraformaldehyde solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713-S</td>
			<td></td>
		</tr>
		<tr>
			<td>25 ml pipettes</td>
			<td>Fisher Scientific</td>
			<td>431032</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G needle</td>
			<td>Exel</td>
			<td>26437</td>
			<td></td>
		</tr>
		<tr>
			<td>3ml luer lock syringe</td>
			<td>Fisher Scientific</td>
			<td>14955457</td>
			<td></td>
		</tr>
		<tr>
			<td>41 x 41 x 8 mm polystyrene weigh dish</td>
			<td>Fisher Scientific</td>
			<td>08-732-112</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conicals</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well, U-bottom assay plate without lid</td>
			<td>Falcon</td>
			<td>353910</td>
			<td></td>
		</tr>
		<tr>
			<td>Amine reactive beads</td>
			<td>ThermoFisher</td>
			<td>A10628</td>
			<td></td>
		</tr>
		<tr>
			<td>Analysis software</td>
			<td>FlowJo</td>
			<td>v 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rat and anti-hamster Ig kappa bead set</td>
			<td>BD Biosciences</td>
			<td>552845</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Jackson Laboratory</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>Invitrogen</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter slides</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>Compensation beads</td>
			<td>ThermoFisher</td>
			<td>01-1111-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Count beads</td>
			<td>ThermoFisher</td>
			<td>01-1234-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-123</td>
			<td></td>
		</tr>
		<tr>
			<td>Digestion enzyme</td>
			<td>Sigma Aldrich</td>
			<td>5401020001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>National Optical and Scientific Instruments, Inc.</td>
			<td>DC3-420T</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissociation tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-334</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic dissociator</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-937</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Diva software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fc block, rat anti-mouse CD16/CD32</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Integra Miltex</td>
			<td>17035X</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow buffer</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow tubes</td>
			<td>Falcon</td>
			<td>352008</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamster anti-mouse CD11c BV421</td>
			<td>BD Biosciences</td>
			<td>562782</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td>Fisher Scientific</td>
			<td>07-210-123</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead dye</td>
			<td>ThermoFisher</td>
			<td>65-0866-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysing solution, 10x solution</td>
			<td>BD Biosciences</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro titer tube and rack</td>
			<td>Fisher Scientific</td>
			<td>02-681-380</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-mouse CD64 PE</td>
			<td>BioLegend</td>
			<td>139304</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-mouse NK1.1 PECF594</td>
			<td>BD Biosciences</td>
			<td>562864</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 pipet tips</td>
			<td>Denville Scientific</td>
			<td>P2404</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 pipettor</td>
			<td>Gilson</td>
			<td>FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>P2 pipet tips</td>
			<td>eppendorf</td>
			<td>22491806</td>
			<td></td>
		</tr>
		<tr>
			<td>P2 pipettor</td>
			<td>Gilson</td>
			<td>FA10001M</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 pipettor</td>
			<td>Gilson</td>
			<td>FA10003M</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 pipet tips</td>
			<td>Denville Scientific</td>
			<td>P2401</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 pipettor</td>
			<td>Gilson</td>
			<td>FA10005M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet man</td>
			<td>Fisher Scientific</td>
			<td>FB14955202</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse B220 PECF594</td>
			<td>BD Biosciences</td>
			<td>562313</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD11b APC-Cy7</td>
			<td>BD Biosciences</td>
			<td>557657</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD19 AlexaFluor 700</td>
			<td>BD Biosciences</td>
			<td>557958</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD19 PE</td>
			<td>BD Biosciences</td>
			<td>553786</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD4 PECF594</td>
			<td>BD Biosciences</td>
			<td>562314</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD45 FITC</td>
			<td>ThermoFisher</td>
			<td>11-0451-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD45 PE-Cy7</td>
			<td>BD Biosciences</td>
			<td>552848</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD8 PECF594</td>
			<td>BD Biosciences</td>
			<td>562315</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Ly6C</td>
			<td>BD Biosciences</td>
			<td>561085</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Ly6G PECF594</td>
			<td>BD Biosciences</td>
			<td>562700</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse MHC II AlexaFluor 700</td>
			<td>BioLegend</td>
			<td>107622</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Siglec F PECF594</td>
			<td>BD Biosciences</td>
			<td>562757</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Tim4 AlexaFluor647</td>
			<td>BD Biosciences</td>
			<td>564178</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking incubator</td>
			<td>Labnet</td>
			<td>311DS</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cell strainer, 40 mm nylon mesh</td>
			<td>Fisher Scientific</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water, 500 ml</td>
			<td>Gibco</td>
			<td>A12873-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Swinging bucket centrifuge</td>
			<td>eppendorf</td>
			<td>5910 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4% solution</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
	</tbody>
</table>

digestion of whole mouse eyes for multi-parameter flow cytometric analysis of mononuclear phagocytes

Det medfødte immunsystem spiller vigtige roller i okulær patofysiologi, herunder uveitis, diabetisk retinopati og aldersrelateret makuladegeneration. Medfødte immunceller, specielt mononukleare fagocytter, udtrykker overlappende celleoverflademarkører, hvilket gør det til en udfordring at identificere disse populationer. Multiparameterflowcytometry giver mulighed for samtidig, kvantitativ analyse af flere celleoverflademarkører for at differentiere monocytter, makrofager, mikroglia og dendritiske celler i museøjne. Denne protokol beskriver enucleation af hele museøjne, okulær dissektion, fordøjelse i en enkelt celle suspension, og farvning af en enkelt celle suspension for myeloid celle markører. Derudover forklarer vi de korrekte metoder til bestemmelse af spændinger ved hjælp af enkeltfarvekontroller og til afgrænselse af positive porte ved hjælp af fluorescens minus en kontrol. Den største begrænsning af multi-parameter flow cytometry er fraværet af vævsarkitektur. Denne begrænsning kan overvindes ved multi-parameter flow cytometry af individuelle okulære rum eller gratis immunofluorescens farvning. Immunfluorescens er imidlertid begrænset af manglende kvantitativ analyse og reduceret antal fluorophorer på de fleste mikroskoper. Vi beskriver brugen af multi-parametrisk flow cytometri til at give meget kvantitativ analyse af mononukleare fagocytter i laser-induceret choroidal neovascularization. Derudover kan multiparameterflowcytometry bruges til identifikation af makrofagundersæt, skæbnetilknytning og cellesortering til transskriptionomik eller proteomiske undersøgelser.

Figur 1 viste ukompenserede frekvens histogrammer for SCC'er og celler til den røde laser: Alexa647, Alexa700 og APC-Cy7. I figur 1A, den magenta linje afbildet toppen af SCC for Alexa647, mens cyan linje viste den påtænkte 0,5 Log forskel. Bemærk, at toppen i Alexa700 og APC-Cy7 var til venstre (mindre lyse) af cyan linje. Bemærk også, at cellerne alle var til venstre for magentalinjen. Alle celler til højre for SCC-toppen blev ikke kompenseret. Specifik...

Watch this Scientific Journal Video about Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes at JoVE.com

Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes

Fordøjelse af hele museøjne til multiparameterflow cytometrisk analyse af mononukleare fagocytter

Denne protokol giver en metode til at fordøje hele øjne i en enkelt celle suspension med henblik på multi-parameter flow cytometrisk analyse med henblik på at identificere specifikke okulære mononukleare phagocytiske populationer, herunder monocytter, mikroglia, makrofager, og dendritiske celler.

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. ...

Research

JoVE Journal

Immunology and Infection

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Flowcytometrisk Isolering af primære murine type II alveolære epitelceller for Funktionelle og molekylære studier

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Whole-dyr Imaging og Flowcytometrisk Teknikker til analyse af antigen-specifikke CD8 + T-celle responser efter Nanopartikel Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Isolering og flowcytometrisk analyse af immunceller fra iskæmisk Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Isolering og flowcytometrianalyse gliom-infiltrerende perifere mononukleære blodceller

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

A Simple flowcytometriske metode til at måle glukoseoptagelse og Glucose Transporter Udtryk for Monocyt undergrupper i fuldblod

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometri af partikelbundet Bet v 1 Allergen i PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

Flowcytometrisk analyse of Natural Killer Cell lytisk aktivitet i humant fuldblod

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

Isolering og flowcytometrisk analyse for Human endocervikale Gamma Delta T-celler

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Karakterisering af humane monocyt delmængder af fuldblod Flow flowcytometri analyse

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Fordøjelsen af Murine leveren en Flow Cytometric analyse af lymfatisk Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...