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Rask og spesifikk Immunomagnetic isolasjon av musen primære Oligodendrocytes

Published: May 21, 2018
doi:
10.3791/57543
, ,
1Program in Molecular and Cellular Biology,State University of New York Downstate Medical Center, 2Department of Neurology and Rehabilitation,University of Illinois at Chicago

Summary

Vi beskriver immunomagnetic isolasjon av hovedknappen på musen oligodendrocytes, som gir rask og spesifikk isolering av celler for i vitro kultur.

Abstract

Effektiv og robust isolasjon og kultur av primære oligodendrocytes (OLs) er et verdifullt verktøy for i vitro studier av utviklingen av oligodendroglia i tillegg til biologi av demyeliniserende sykdommer som multippel sklerose og Pelizaeus-Merzbacher-lignende sykdom (PMLD). Her presenterer vi en enkel og effektiv utvalg metode for immunomagnetic isolasjon av fase tre O4+ preoligodendrocytes celler fra neonatal mus pups. Siden umodne OL utgjør mer enn 80% av gnager-hjerne hvit saken i postnatal dag 7 (P7) denne isolasjon metoden ikke bare sikrer høy mobil ytelse, men også spesifikke isolasjon OLS allerede forpliktet til oligodendroglial slektslinje, redusere den muligheten for å isolere forurensende cellene som astrocyttene og andre fra musen hjernen. Denne metoden er en modifikasjon av teknikkene rapportert tidligere, og gir oligodendrocyte forberedelse renhet over 80% på omtrent 4 h.

Introduction

Oligodendrocytes (OLs) er de myelinating cellene i sentralnervesystemet (CNS)1. Isolasjon og kultur av primære oligodendrocytes i et strengt regulert miljø er et verdifullt verktøy for i vitro studier av utviklingen av oligodendroglia i tillegg til biologi av demyeliniserende sykdommer som multippel sklerose2 . Dette krever en effektiv og robust oligodendrocyte isolasjon og kultur metode3. I denne studien tok vi fordel av uttrykk for en særegen oligodendrocyte celle overflaten markør å implementere en modifisert isolasjon teknikk som er rask og spesifikk.

Fire forskjellige stadier av oligodendrocyte modning har blitt identifisert, hver preget av uttrykk for karakteristiske celle overflate markører for hver utviklingsstadiet (figur 1). Stikkordmerker cellen overflaten kan bli gjenkjent av spesifikke antistoffer4,5, og kan brukes til å isolere OLs på bestemte stadier. I den første fasen har oligodendrocyte forløper celler (OPCs) kapasitet til å spre, overføre og spesielt Ekspress blodplater-avledet vekst faktor reseptor (PDGF-Rα)6, ganglioside A2B5, proteoglycan NG27,8 , polysialic syre-nevrale celle vedheft molekyl9 og fatty-acid-bindende protein 7 (FABP7)10. OPCs har bipolar morfologi med noen korte prosesser kommer fra de motsatte polakkene av celle som er karakteristisk for neural forløper celler11.

Figure 1
Figur 1: uttrykk for cellen overflate markører under musen oligodendrocyte utviklingen. OLs celle overflate markører som A2B5, GalC (O1), NG2, O4 og PDGF-Rα kan brukes til å isolere spesielt oligodendrocytes på bestemte utviklingsstadiet ved hjelp av spesifikke antistoffer.   Klikk her for å se en større versjon av dette tallet.

I den andre fasen, OPCs gi opphav til preoligodendrocytes og express på cellemembranen ikke bare OPC markører, men også sulfatide (en sulfated galactolipid) anerkjent av O4 antistoff12,13, og i GPR17 protein14, som vedvarer til umodne oligodendrocyte (OL) scenen. På dette stadiet utvide preoligodendrocytes multipolar kort prosesser. Preoligodendrocytes er den store OL-scenen postnatal dag 2 (P2) i cerebral hvit saken rotte og mus med en liten befolkning umodne OLs15.

I den tredje fasen fortsatt umodne OLs å uttrykke O4, mister uttrykk for A2B5 og NG2 og begynne å uttrykke galactocerebroside C16. På dette stadiet, OLs er forpliktet til oligodendroglial slektslinje og bli etter mitotisk celler med lang ramified grener17,18. Umodne OL utgjør mer enn 80% av gnager hvit saken på P7 og nå første MBP+ cellene er observert15,19,20,21. Derfor kunne isolasjon OLS på P7 sikre høy mobil ytelse.

I den siste og fjerde fasen av OL utvikling uttrykke modne OLs myelinating proteiner (myelin basic protein (MBP), proteolipid protein (PLP), myelin forbundet glykoprotein (MAG) og myelin oligodendrocyte glykoprotein (MOG)22,23 ,24,25,26. På dette stadiet, modne OLs utvide membraner som skjemaet kompakt enwrapping hylser rundt axons og kan myelinate. Dette sammenfaller med observasjon at i rotte og mus hjernen, MBP+ celler blir stadig rike på P1419,20,21.

Siden første isolering av oligodendrocyte av Fewster og kolleger i 196727, har flere metoder for isolering av OLs fra gnagere CNS implementert inkludert immunopanning28,29,30, fluorescens-aktivert celle sortering (FACS) utnytte cellen overflaten-spesifikke antigener28,31, differensial gradient sentrifugering32,33,34,35 og risting metode basert på differensial etterlevelse av ulike CNS glia36,37. Eksisterende kultur metoder har imidlertid begrensninger, særlig i forhold til renhet, avkastning og tiden det tar å utføre prosedyrer38. Mer effektiv isolasjon metoder for oligodendrocytes er derfor nødvendig.

I dette papiret, presenterer vi en enkel og effektiv utvalg metode for immunomagnetic isolasjon av scene tre O4+ preoligodendrocytes celler fra neonatal mus pups. Denne metoden er en modifikasjon av teknikkene rapportert av Emery et al. 39 og Dincman et al. 40 og gir en oligodendrocyte forberedelse renhet over 80% på omtrent 4 h.

Protocol

Musene som brukes i denne studien var stelt i henhold til retningslinjene i protokollnummeret SUNY Downstate Medical Center divisjon av laboratoriet dyr ressurser (DLAR) 15-10492. Merk: Primære oligodendrocytes ble isolert fra neonatal (P5-P7 vill-type C57Bl/6N) mus. På dette stadiet utgjør umodne OLs mer enn 80% av gnager hvit saken sikrer høy mobil ytelse. Alle buffer og reagens komposisjoner er tilgjengelig på slutten av Tabellen for materiale. <p class="jove_title…

Representative Results

Formålet med denne studien var å etablere en økt isolering metode for O4+ hovedknappen på musen oligodendrocytes som krever minst mulig manipulering av målcellene. Hele prosedyren fra eutanasi av valpene til plating cellene i coverslips tar ca 4 h og dataene som vises her representerer tre uavhengige eksperimenter. Etter vev dissosiasjon var gjennomsnittlig 4,3 ± 0.46 x 107 celler isolert for hver uavhengige eksperimentet med en levedyktigheten til 91% ± 5.6. …

Discussion

I dette innlegget presenterer vi en metode for effektiv isolasjon av høyrenset umodne musen oligodendrocyte kulturer. I forhold til tidligere publiserte protokoller39,40, gitt denne metoden en høyere renhet med et mye lavere nivå av GFAP-positive astrocyttene og en svært lav prosentandel av andre ikke-preget celler. Det er viktig å påpeke at disse er umodne OLs allerede forpliktet seg til den oligodendroglial avstamning. Dermed ville disse cellene ikke vær…

Declarações

The authors have nothing to disclose.

Acknowledgements

Denne studien ble støttet av tilskudd fra det nasjonal multippel sklerose samfunnet (RG4591A1/2) og National Institutes of Health (R03NS06740402). Forfatterne takker Dr. Ivan Hernandez og hans lab medlemmer for å gi laboratoriet plass, utstyr og råd.

Materials

10ml serological pipets Fisher Scientific 13-676-10J
10ml syringe Luer-Loc tip BD, Becton Dickinson 309604
15ml conical tubes Falcon 352097
24-well tissue culture plates Falcon 353935
40µm cell strainer Fisher Scientific 22368547
50ml conical tubes Falcon 352098
5ml serological pipets Fisher Scientific 13-676-10H
60mm tissue culture plates Falcon 353002
70µm cell strainer Fisher Scientific 22363548
Alexa Fluor 488 goat anti-mouse IgG (H+L) secondary antibody Invitrogen A11001
Alexa Fluor 488 goat anti-rabbit IgM (H+L) secondary antibody Invitrogen A21042
Alexa Fluor 488 goat anti-rabbit IgM (H+L) secondary antibody Invitrogen A11008
Alexa Fluor 594 goat anti-chicken IgG (H+L) secondary antibody Invitrogen A11042
Anti-O4 beads- Anti-O4MicroBeads Miltenyi Biotec 130-094-543
Apo-Transferrin human Sigma T1147
Autofil complete bottle top filter assembly, 0.22um filter, 250ml USA Scientific 6032-1101
Autofil complete bottle top filter assembly, 0.22um filter, 250ml USA Scientific 6032-1102
B27 Supplement Invitrogen 17504-044
Boric acid Sigma B7660
Bovine Growth Serum (BGS) GE Healthcare Life Sciences SH30541.03
BSA Fisher Scientific BP-1600-100
CNTF Peprotech 450-50
d-Biotin Sigma B4639
Desoxyribonuclease I (DNAse I) Worthington LS002007
EDTA Fisher Scientific S311
Epifluorescence microscope with an Olympus DP70 camera Olympus Bx51
Feather disposable scalpels Andwin Scientific EF7281C
Forskolin Sigma F6886
German glass coverslips, #1 thickness, 12mm diameter round NeuVitro GG-12-oz
GFAP antibody Aves GFAP
Glucose Fisher Scientific D16-1
GlutaMAX Invitrogen 35050-61
Insulin Invitrogen 12585-014
Magnetic separator stand – MACS multistand Miltenyi Biotec 130-042-303
Magnetic separator-MiniMACS separator Miltenyi Biotec 130-042-302
Millex PES 0.22µm filter unit Millipore SLG033RS
Mounting media- Prolong Gold with DAPI Thermo Fisher P36930
N-acetyl-cysteine (NAC) Sigma A8199
Natural mouse laminin Invitrogen 23017-015
Neurobasal Medium A Invitrogen 10888-022
Neurotrophin-3 (NT-3) Peprotech 450-03
NG2 antibody Millipore AB5320
Papain Worthington LS003126
PBS without Ca2+ and Mg2+ Sigma D5652
PDGF Peprotech 100-13A
Petri dishes Falcon 351029
Poly-D-Lysine Sigma P6407
Primocin Invivogen ant-pm-2
Progesterone Sigma P8783
Putrescine Sigma P5780
Selection column-LS columns Miltenyi Biotec 130-042-401
Sodium Selenite Sigma S5261
Trace elements B Corning 25-000-CI
Triiodothyronine (T3) Sigma T6397
Triton-X Sigma T8787
Trypan Blue Solution Corning 25-900-CI
Tween 20 Sigma P1379
B27NBMA 487.75 mL Neurobasal Medium A; 10 mL B27 Supplement; 1 mL Primocin; 1.25 mL Glutamax; Filter sterilize and store at 4 °C until use.
B27NBMA + 10% BGS 27 mL B27NBMA; 3 mL Bovine growth serum
CNTF solution stock (10 µg/ml; 1000X) Order from Peprotech (450-50). Make up at 0.1 to 1 mg/ml according to Manufacturer’s instruction (may vary from lot to lot) in buffer (e.g. DPBS + 0.2% BSA). Store at -80 °C.
Working solution (10 µg/ml, 1000X)
1. Make on 0.2% BSA (Fisher scientific BP-1600-100) in DPBS solution and filter sterilize.
2. Dilute master stock aliquot to 10µg/ml in sterile, chilled 0.2% BSA/DPBS.
3. Aliquot (20µl/tube) and snap freeze in liquid nitrogen.
4. Store aliquots at -80 °C.
d-Biotin stock solution (50 µg/ml; 5000X) Resuspend d-Biotin (Sigma-B4639) in double-distilled H2O at 50 µg/ml (e.g. 2.5 mg in 50 ml of ddH2O). Resuspension might take fair amount of agitation/vortexing, or mild warming briefly at 37°C. If the d-Biotin still will not solubilize, it is fine to make up a less concentrated (e.g. 10µg/ml), and to add a higher volume to the B27NBMA (1/1000), instead of 1/5000). Store at 4°C.
DNase I stock solution 1. Dissolve at 12,500 U Deoxyribonuclease I / ml in HBSS chilled on ice.
2. Filter sterilize on ice
3. Aliquot at 200 µl and freeze overnight at -20°C.
4. Store aliquots at -20 to -30°C.
Dulbecco’s Phosphate Buffered Saline (w/o Ca2+ and Mg2+) Dissolve pouch in 1 Liter of water to yield 1 liter of medium at 9.6 grams of powder per liter of medium. Store at 2-8 °C.
Forskolin stock solution (4.2 mg/ml; 1000X) Add 1 ml of sterile DMSO to 50 mg Forskolin in bottle (Sigma-F6886) and pipette until resuspended. Transfer to a 15 ml centrifuge tube and add 11 ml of sterile DMSO to bring to 4.2 mg/ml. Aliquot (e.g. 20 µl) and store at -20°C.
Hank’s balanced salts (HBSS) (Sigma 1. Measure 900 ml of water (temperature 15-20 °C) in a cylinder and stir gently.
2. Add the power and stir until dissolved.
3. Rinse original package with a small amount of water to remove all traces of the powder.
4. Add to the solution in step 2.
5. Add 0.35 gr of sodium bicarbonate (7.5% w/v) for each liter of final volume.
6. Keep stirring until dissolved.
7. Adjust the pH of the buffer while stirring to 0.1-0.3 units below pH= 7.4 since it may rise during filtration. The use of 1N HCl or 1N NaOH is recommended to adjust the pH.
8. Add additional water to bring the final volume to 1L.
9. Sterilize by filtration using a membrane with a porosity of 0.22 microns.
10. Store at 2-8 °C.
Insulin stock solution (4000 µg/ml) Thaw the bottle and aliquot 25 µl per microcentrifuge tube and store at -20°C.
Laminin solution Slowly thaw laminin in the cold (2°C to 8°C) to avoid gel formation. Then, aliquot into polypropylene tubes. Store at 5° C to -20° C in aliquots (e.g. 20 µl) and do not freeze/thaw repeatedly. Laminin may be stored at these temperatures for up to six months.
Magnetic Cell Sorting (MCS) Buffer Prepare the solution containing phosphate-buffered saline (PBS), pH 7.2, and 0.5% bovine serum albumin (BSA), 0.5 mM EDTA, 5µg/ml Insulin, 1 g/L Glucose. Sterilize and degas by filtration the buffer by passing it through a 0.22 µm Millex filter. Store the buffer at 4°C until use
N-Acetyl-L-cysteine (NAC) stock solution (5mg/ml; 1000X) Dissolve N-Acetyl-L-cysteine (Sigma-A8199) at 5 mg/ml in DMEM (e.g. 50 mg NAC in 10 ml B27NBMA). Filter sterilize and aliquot (e.g. 20 µl). Store at -20°C.
NT3 stock solution (1 µg/ml; 1000X) Master stock:
Order from Peprotech (450-03). Make up at 0.1 to 1 mg/ml according to manufacturer’s instructions (may vary from lot to lot), in buffer (e.g. DPBS + 0.2% BSA). Store at -80°C.

Working stock (1µg/ml; 1000X):
1. Make on 0.2% BSA in DPBS solution and filter sterilize.
2. Dilute master stock aliquot to 1 µg/ml in sterile, chilled 0.2% BSA/DPBS.
3. Aliquot (e.g. 20µl/tube) and snap freeze in liquid nitrogen.
4. Store aliquots at -80°C.
PDGF stock solution (10 µg/ml; 1000X) Master stock:
Order from Peprotech (100-13A). Make up at 0.1 to 1 mg/ml according to manufacturer’s instructions (may vary from lot to lot) in buffer (e.g. DPBS) + 0.2% BSA). Store at -80°C.

Working stock (1µg/ml; 1000X):
1. Make on 0.2% BSA in DPBS solution and filter sterilize.
2. Dilute master stock aliquot to 1µg/ml in sterile, chilled 0.2% BSA/DPBS.
3. Aliquot (e.g. 20µl/tube) and snap freeze in liquid nitrogen.
4. Store aliquots at -80°C.
Poly-D-lysine (1mg/ml; 100X) Resuspend poly-D-lysine, molecular weight 70-150 kD (Sigma P6407) at 0.5mg/ml in 0.15M boric acid pH 8.4 (e.g. 50mg in 50ml borate buffer). Filter sterilize and aliquot (e.g. 100µl/tube). Store at -20°C. Prior to use, dilute the 100X stock (1mg/ml) to 50 µg/ml in sterile water.
Oligodendrocyte proliferation media see Supplementary Table 1
Oligodendrocyte differentiation media see Supplementary Table 1
Sato supplement (100X) see Supplementary Table 1
References: the list of reagents and recipes were adopted from the protocols previously described by Emery et. al. 2013 (Emery, B. & Dugas, J. C. Purification of oligodendrocyte lineage cells from mouse cortices by immunopanning. Cold Spring Harb Protoc. 2013 (9), 854-868, doi:10.1101/pdb.prot073973, (2013)) and Dincman et. al. (Dincman, T. A., Beare, J. E., Ohri, S. S. & Whittemore, S. R. Isolation of cortical mouse oligodendrocyte precursor cells. J Neurosci Methods. 209 (1), 219-226, doi:10.1016/j.jneumeth.2012.06.017, (2012))
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Tags

Keywords: Immunomagnetic IsolationMouse Primary OligodendrocytesMyelinMyelinationNeural ScienceCell CultureDissociationNeurobasal A MediumBovine Growth SerumDNase IPipetting
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Flores-Obando, R. E., Freidin, M. M., Abrams, C. K. Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes. J. Vis. Exp. (135), e57543, doi:10.3791/57543 (2018).
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