Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Vishal Menon; Ria Thomas; Arun R. Ghale; Christina Reinhard; Jan Pruszak

doi:10.3791/52241

JoVE Journal > Neuroscience

Neurociência

Flowcytometri Protokoller for Surface og intracellulære Antigen Analyser af neurale Cell Typer

Published: December 18, 2014

doi:

10.3791/52241

Vishal Menon, Ria Thomas², Arun R. Ghale³, Christina Reinhard, Jan Pruszak⁴

¹Emmy Noether-Group for Stem Cell Biology, Department of Molecular Embryology, Institute of Anatomy and Cell Biology,University of Freiburg, ²Spemann Graduate School of Biology and Medicine and Faculty of Biology,University of Freiburg, ³School of Life Sciences,Keele University, ⁴Center for Biological Signaling Studies (BIOSS),University of Freiburg

Summary

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

Abstract

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

Introduction

Flowcytometri er blevet grundigt udnyttet i immunologi, hæmatologi og onkologi at definere cellepopulationer via iboende scatter egenskaber, celleoverfladeantigen ekspression og andre fluorescensparametre ^1-3. Vores indsigt i slægt udvikling og sygdom er et resultat at en betydelig grad af den kontinuerlige forfining af denne metode efter dens oprindelige ^4,5 gennemførelse. Øget bevidsthed om den kvantitative og den samlede analytiske potentiale flowcytometri nylig tilskyndet sin mere udbredt anvendelse i stamcelleforskning og kan gøre det muligt på samme måde dybe fremskridt i en kortere tidsramme ^6. Imidlertid har anvendelsen af flowcytometri til specifikt at analysere og isolere neurale populationer længe været opfattet som udfordrende. I modsætning til hæmatopoietiske celler, som naturligt findes i suspension, er neurale celletyper typisk høstet fra overdrevent komplekse kilder, der kan omfatte glia og forskellige other omkringliggende celler samt et indviklet netværk af proces-bærende neuroner. Følgelig har neurobiologi endnu at gennemføre alsidighed flowcytometri til dets fuldstændige potentiale i daglige forskning rutiner. Men så længe som en levedygtig enkelt cellesuspension kan genereres (og protokoller er blevet udviklet og optimeret til dette formål ^7), flowcytometri og fluorescens-aktiveret cellesortering (FACS) kan betragtes som et værdifuldt element i den analytiske repertoire i neurobiologi ^8-11.

. Figur 1. Princippet om flowcytometrisk analyse og komponenter i et flowcytometer flowcytometre omfatter tre vigtigste systemer: fluidik, optik og elektronik. En strømlinet flow af celler i suspension (fremstillet ud fra primært væv eller in vitro kultur) opnås ved hylsteret bomuld, lid via hydrodynamisk fokusering, begrænser prøven til dens centrum kerne. De optiske dele er sammensat af lasere, der belyser strømmen af celler og optiske filtre, der dirigerer signalet til passende detektorer. De lyssignaler detekteres konverteres til elektroniske signaler, derefter behandles af en computer og visualiseret på en skærm til dataanalyse og gating. Klik her for at se en større udgave af dette tal.

Brugere af flowcytometriske metoder profit fra mindst en grundlæggende forståelse af de underliggende fundamentale, herunder en cytometer byggesten (til gennemgang se ^12,13; se også figur 1). En laserstråle skærer med en hydrodynamisk fokuseret strømningsteknisk strøm, der indeholder de celler i suspension, som igen passerer gennem laserstrålen i "enkelt fil« ene efter den anden. Den interceptipå en celle (eller en anden partikel, for den sags skyld) med laseren resulterer i spredning af lys fra denne forespørgsel punkt. Spredt lys kan detekteres i forlængelse af laser retning (forward scatter forbundet med størrelsen af partiklen), samt vinkelret på dens retning (side scatter, afspejler granulosity af partiklen / celle). Disse førnævnte scatter egenskaber ikke kræver specifik mærkning, hvilket er grunden til en umærket prøve (eller også celleaffald, luftbobler, etc.) vil generere et signal (begivenhed) på bivariate forward scatter versus side scatter plot almindeligvis anvendes til indledende gating. Ved anvendelse af de passende lasere og filtre specifikke for den tilsvarende excitations- og emissionsspektre, kan en celle analyseres for positivitet, lysstyrkeniveau, eller fravær af fluorescerende markører. Størstedelen af flowcytometrisk ansøgninger har fokuseret på karakterisering via celleoverfladeantigener. I modsætning til den hæmatopoietiske lineage har neurale afstamning forblevet mindre udstrækning defineret i henhold til overfladen epitop ekspressionsmønstre ^5. En fordel ved at udnytte overfladeantigener er, at levende celler kan underkastes cellesortering paradigmer såsom FACS. I modsætning hertil intracellulær antigenfarvning kræver fiksering og permeabilisering skridt til at mediere epitop-antistof-interaktion, er til hinder for efterfølgende anvendelser, der kræver levedygtige celler. Notatet, stadig Herved vil for mange kvantitative analyser ¹⁴ og downstream analyser for RNA og protein-ekspression ^15. Hæmatologi, immunologi og onkologi har ofte brugt mere end et dusin markører sammen for at definere bestemte subpopulationer ^16. Derudover kan masse cytometri eller CyTOF nu bruges til at analysere op til 30 parametre samtidigt ^17,18.

For neurale stamceller applikationer samt primære kulturer ^14,19,20 heterogenitet celler ivitro er et almindeligt fænomen ^21-23. Cellerne ikke repræsenterer målgruppen af interesse indebærer en potentielt forstyrrende element for eksperimentel udlæsning ^24,25. Bekvemt, de forskellige cellulære delmængder stede i en heterogen cellesuspension bære forskellige (kendte eller endnu tydes) antigenekspression profiler, som kan anvendes til at definere de forskellige befolkningsgrupper. Flowcytometri kan derfor spille en afgørende rolle i løsningen af cellulær heterogenitet og dermed lette biomedicinske anvendelser (in vitro-assays, celleterapi) og optimere kvantitativ udlæsning ved at fokusere på de mest relevante delmængde ^24,26. Forskellige overflade antigen kombinationer er blevet identificeret i løbet af de sidste par år for at tillade kvantificering og isolering af specifikke neurale celletyper. Dette omfatter CD133 til berigelse af neurale stamceller ^27, en kombination af CD15 / CD24 / CD29 overfladeantigener til isolering af NSC, differentieringTed neuron og neurale crest celler ²⁸ eller CD15 / CD24 / CD44 / CD184 / CD271 at isolere neurale og gliale delmængder ^25, blandt andre signaturer ^29,30. Beyond neuroner, gliale markører indbefatter A2B5 ^31, CD44 ^25. NG2 ³² og GLAST ^33. En nylig publikation har udnyttet midthjernen gulvpladen forløber markør CORIN ^34,35 at berige for dopaminerge prækursorer i Parkinson-celle transplantation paradigmer ^36. CD-molekyler er ikke kun markører, men funktionelt relevante mediatorer af celle-celle-interaktioner og i en celles evne til at reagere på signaler fra ekstracellulære matrixmolekyler og vækstfaktorer ^37. En strategi om yderligere at styrke det arsenal af kombinatoriske CD-antigener til at karakterisere neurale afstamning udvikling er at bruge kendte intracellulære markører til at screene for og definere CD antigen kombinationer for en bestemt celletype af interesse. Vi har for nylig udnyttet en sådan tilgang og identificeret CD49f ^– / CD200 ^høje kombinatoriske udtryk mønstre som en ny tilgang til berigelse af neuronale delmængder fra neuralt differentierede induceret pluripotente stamceller dyrkningssystemer ^38. Her inkluderer vi og diskuterer sidstnævnte protokol (og valgfrie variationer deraf), hvor overfladen farvning og intracellulær farvning kan anvendes samtidig til at definere neurale celle subpopulationer ved flowcytometri.

Figur 2. Blokdiagram over forsøgsprotokol muligheder. Figuren viser en skematisk gengivelse af de vigtigste trin i protokollen. Valgfri trin (CFSE farvestof eller intracellulære antigen mærkning) er angivet med lysegrå bokse. Efter høstning, er det vigtigt at vurdere levedygtigheden og celleantal af neurale cellesuspensioner før celleoverfladefarvning. Positive somsåvel som negative kontroller bør indgå i tillæg til de prøver af interesse. Prøverne kan analyseres ved flowcytometrisk analyse og / eller anvendes i celle sortering paradigmer. Klik her for at se en større udgave af dette tal.

Mens vi tidligere har brugt primære antistof i kombination med sekundært antistof til intracellulær farvning ³⁸ vi nu indføre ikke-kovalent mærkning af det primære antistof via fluorescerende Fab-fragmenter (Zenon mærkning) som en lille variation, hvorved trinnene cellemanipulationsniveauet ^39. Som et yderligere eksempel på protokollens alsidighed, vi ansætter en valgfri mærkning af en eksperimentel delmængde ved carboxyfluorescein succinimidylester (CFSE) før overflade antigen farvning. Sådanne CFSE pre-mærkning muliggør umiddelbar direkte sammenligning af to cellelinier eller eksperimentelle betingelser (CFSE-mærket vs. umærket) inden for en enkelt prøve rør, reducere variansen eller subtile forskelle i inkubationstid og spare antistof. CFSE er en etableret fluorescerende farvestof, der er almindeligt anvendt til cellesporing ^40, i proliferation ^41,42 og stregkodesystem eksperimenter ^43,44. Endelig mens de faktiske sorterings- trin (FACS, immunomagnetisk celle separation eller immunopanning) er ikke en del af denne protokol, i princippet de høst- og mærkningsprocedurer her beskrevne gøre udbytte prøver, der kan udsættes til overfladen antigen eller intracellulære mærkning baseret sortering applikationer ^{15 25,28.}

Med denne artikel, tilstræber vi at: sammenfatte en levedygtig overfladeantigen farvningsprotokol ^25,28, opsummere en protokol til påvisning af intracellulære mål samt kombineret overflade og intracellulære antigen analyse ^38, præsentere en intracellulær CFSE farvestof etikettering trin ^41,45 som en eksperimentel mulighed for comkomparativ analyse af neurale cellepopulationer, og opsummere tilgange til flowcytometrisk analyse (passende kontrol ^13,46, gating strategi og datapræsentation ^47).

Protocol

1. Neural Cell Høst Vurdering mikroskop: Før iværksættelse af et eksperiment, kontrollere status for kulturen med lyse-felt eller fasekontrastmikroskopi. BEMÆRK: Mens primær neurale væv fremstillet af dissektioner er i princippet lige så modtagelige for flowcytometrianalyse 14,28, bemærk venligst, at protokollen fokuserer på celler fra in vitro neurale cellekulturer. Høst celler 7: Skålen / kolbe af adhærente celler med M…

Representative Results

Protokollen præsenteres her giver mulighed for alsidig eksperimentelle tilgange (Figur 2). I sin korteste version (trin 1, 3 og 6), kan det betragtes som en guide for simpel farvning af overfladeantigener. I sin mere kompleks form, kan en række co-mærkning paradigmer med en række af intracellulære antigener forfølges (valgfrit trin 2 og / eller 4 til 5). Desuden trin CFSE-mærkning eksemplificerer dens anvendeligh…

Discussion

Protokollen præsenteres her er veletableret for neurale cellekulturer afledt fra humane stamceller, men kan også anvendes til andre neurale celle kilder, herunder primært væv eller neurale cellelinier. Ud over embryonale kilder, kan neurale stam- eller progenitorceller ekstraheres fra den neurogene områder af voksne hjerne ^27. Desuden flowcytometri og FACS kan udnyttes til at kvantificere, analysere og isolere forskellige cellepopulationer, herunder modne neuroner ^54, astroglia ^33, …

Declarações

The authors have nothing to disclose.

Acknowledgements

Our research program is funded through the Emmy Noether-Program of the German Research Foundation (DFG), grant PR1132/3-1. Further support by the Müller-Fahnenberg Foundation of the University of Freiburg is gratefully acknowledged. This study was supported in part by the Excellence Initiative of the German Research Foundation (GSC-4, Spemann Graduate School).

Materials

DMEM/F12 (1:1) (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12)	Life Technologies	11330057	www.lifetechnologies.com
DPBS without Ca²⁺ Mg²⁺	Life Technologies	14190169	www.lifetechnologies.com
Fetal bovine serum, qualified, E.U.-approved, South America origin (FBS)	Life Technologies	10270-106	www.lifetechnologies.com
MEM Non-essential amino acids (100 x)	Life Technologies	11140035	www.lifetechnologies.com
TrypLE Express	Life Technologies	12604013	www.lifetechnologies.com
Trypan blue solution, 0.4%	Life Technologies	15250061	www.lifetechnologies.com
Paraformaldehyde	Carl Roth	335.3	www.carlroth.com
Bovine serum albumin (BSA) Fraction V	PAA Laboratories, Coelbe	K41-001	www.gelifesciences.com
Tween-20 Detergent	Calbiochem	655205	www.merckmillipore.de
Carboxyfluorescein succinimidyl ester (CFSE)	eBioscience	65-0850-84	www.ebioscience.com
DMSO	AppliChem	A1584	www.applichem.com
Bottle top filters express plus 0.22 µm, 250 ml	Millipore	SCGPU02RE	www.millipore.com
Cell culture treated flasks (T 25)	NUNC	156367	www.thermoscientific.com
Cell culture treated flasks (T 75)	NUNC	156499	www.thermoscientific.com
Conical tubes (15 ml)	Greiner Bio-One	188271	www.greinerbioone.com
Conical tubes (50 ml)	Greiner Bio-One	227261	www.greinerbioone.com
Pasteur pipet, glass (150 mm)	STEIN Labortechnik, Remchingen	S03710150	www.stein-labortechnik.de
Pipet tips (0.1-10 µl)	Corning	4125	www.corning.com
Pipet tips (1-200 µl)	Corning	4126	www.corning.com
Pipet tips (100-1000 µl)	Corning	4129	www.corning.com
Serological pipets, 5 ml	Corning	4051	www.corning.com
Serological pipets, 10 ml	Corning	4101	www.corning.com
Serological pipets, 25 ml	Corning	4251	www.corning.com
Microcentrifuge tubes (0.5 ml)	Sarstedt	72,699	www.sarstedt.com
Microcentrifuge tubes (1.5 ml)	Greiner Bio-One	616201	www.greinerbioone.com
Microcentrifuge tubes (2.0 ml)	Sarstedt	72,695,500	www.sarstedt.com
Anti-Human CD24 APC monoclonal antibody	eBioscience	17-0247-42	www.ebioscience.com Working dilution 1:50
Anti-Human CD54 PE monoclonal antibody	eBioscience	12-0549-42	www.ebioscience.com Working dilution 1:50
Neuronal Class III β-Tubulin (Tuj1) polyclonal antibody	Covance	PRB-435P	www.covance.com Working dilution 1:2000
Alexa Fluor 488 Donkey anti Rabbit	Life Technologies	A21206	www.lifetechnologies.com Working dilution 1:2000
Zenon® Fluorescein Rabbit IgG Labeling Kit	Life Technologies	Z-25342	www.lifetechnologies.com

Neubauer-Improved counting chamber	Marienfeld	0640010	www.marienfeld-superior.com
Vortex	Scientific Industries	G560E	www.scientificindustries.com
Thermomixer comfort	Eppendorf	5355 000.001	www.eppendorf.com
Accuri C6 flow cytometer	Becton Dickinson (BD)	653118	www.bdbiosciences.com/instruments/accuri
Microcentrifuge refrigerated, PerfectSpin 24 R	Peqlab	91-PSPIN-24R	www.peqlab.de
Orbital shaker, Unimax 1010	Heidolph	543-12310-00	www.heidolph-instruments.de
Centrifuge refrigerated, Rotanta 96 RC	Hettich	4480-50	www.hettichlab.com
Class II Biological safety cabinet Safe 2020	Thermo Scientific	51026640	www.thermoscientific.com
CO₂ Incubator, Heracell 240i	Thermo Scientific	51026331	www.thermoscientific.com
Vacuum system, Vacusafe comfort	Integra Biosciences	158320	www.integra-biosciences.de
Microscope, Axiovert 40 CFL	Zeiss	451212	www.zeiss.de
Pipet controller, accu-jet pro	Brand	26303	www.brand.de
Micropipet, Pipetman neo P20N (2-20 µl)	Gilson	F144563	www.gilson.com
Micropipet, Pipetman neo P200N (20-200 µl)	Gilson	F144565	www.gilson.com
Micropipet, Pipetman neo P1000N (100-1000 µl)	Gilson	F144566	www.gilson.com

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Menon, V., Thomas, R., Ghale, A. R., Reinhard, C., Pruszak, J. Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types. J. Vis. Exp. (94), e52241, doi:10.3791/52241 (2014).