Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

Rafael E. Flores-Obando; Mona M. Freidin; Charles K. Abrams

doi:10.3791/57543

JoVE Journal > Neuroscience

신경과학

Hurtig og specifik Immunomagnetic Isolation af musen primære Oligodendrocytes

Published: May 21, 2018

doi:

10.3791/57543

Rafael E. Flores-Obando, Mona M. Freidin, Charles K. Abrams

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 isolation af primære mus oligodendrocytes, som giver mulighed for hurtig og specifik isolering af celler til in vitro- kultur.

Abstract

Effektiv og robust isolation og kultur af primære oligodendrocytes (OLs) er et værdifuldt redskab til in vitro- undersøgelse af udviklingen af oligodendroglia samt biologi demyeliniserende sygdomme som sclerose og Pelizaeus-Merzbacher-lignende sygdom (PMLD). Her præsenterer vi en enkel og effektiv udvælgelsesmetode til immunomagnetic isolering af fase tre O4⁺ preoligodendrocytes celler fra neonatal mus unger. Eftersom umodne OL udgør mere end 80% af gnaver-hjernens hvide substans på postnatal dag 7 (P7) denne isolation metode ikke kun sikrer høj cellulære udbytte, men også de specifikke isolering af OLs allerede forpligtet til oligodendroglial slægt, faldende de mulighed for at isolere kontaminerende celler såsom astrocytter og andre celler fra mus hjernen. Denne metode er en ændring af de teknikker, der rapporterede tidligere, og giver oligodendrocyte forberedelse renhed over 80% i ca 4 h.

Introduction

Oligodendrocytes (OLs) er myelinating cellerne i centralnervesystemet (CNS)¹. Isolation og kultur af primære oligodendrocytes i et stramt reguleret miljø er et værdifuldt redskab til in vitro- undersøgelse af udviklingen af oligodendroglia samt biologi demyeliniserende sygdomme som sklerose². Dette kræver en effektiv og robust oligodendrocyte-isolering og kultur metode-³. I denne undersøgelse benyttede vi sig af udtrykket af en karakteristisk oligodendrocyte celle overflade markør til at gennemføre en modificeret isolation teknik, der er hurtig og specifik.

Fire forskellige stadier af oligodendrocyte modning er blevet identificeret, hver karakteriseret ved udtryk for karakteristiske celle overflade markører for hver udviklingsstadiet (figur 1). Disse celle overflade markører kan blive genkendt af specifikke antistoffer⁴^,⁵, og kan bruges til at isolere OLs på konkrete etaper. I den første fase har oligodendrocyte forløber celler (OPCs) kapacitet til at formere sig, overflytte, og specielt express Trombocyt-afledt vækst faktor receptor (PDGF-Rα)⁶, ganglioside A2B5, proteoglycan NG2⁷^,⁸, polysialic syre-neurale celle vedhæftning molekyle⁹ og fede-syre-bindende protein 7 (FABP7)¹⁰. OPCs har bipolar morfologi med par korte processer stammer fra de modsatte poler af cellen organ, som er karakteristisk for neurale forløber celler¹¹.

Figur 1: udtryk for celle celleoverflademarkører under musen oligodendrocyte udviklingen. OLs cellens overflade markører, såsom A2B5, GalC (O1), NG2, O4 og PDGF-Rα kan bruges til specifikt isolere oligodendrocytes på specifikke udviklingsmæssige stadie ved hjælp af specifikke antistoffer. Venligst klik her for at se en større version af dette tal.

I anden fase, OPCs give anledning til preoligodendrocytes og express på cellemembranen OPC markører, men også sulfatide (en sulfated galactolipid) anerkendt af O4 antistof¹²^,¹³, og GPR17 protein¹⁴, som fortsætter indtil stadiet umodne oligodendrocyte (OL). På dette stadium udvide preoligodendrocytes multipolær kort processer. Preoligodendrocytes er den store OL Stadium på postnatal dag 2 (P2) i den cerebrale hvide substans i både rotter og mus med en mindre befolkning af umodne OLs¹⁵.

I den tredje fase fortsat umodne OLs express O4, mister udtryk af A2B5 og NG2 markører og begynde at udtrykke galactocerebroside C¹⁶. På dette stadium, OLs er forpligtet til oligodendroglial slægt og blive post mitotiske celler med lange forgrenet grene¹⁷^,¹⁸. Umodne OL udgør mere end 80% af den gnavere hvide substans på P7 og på dette tidspunkt observeres de første MBP⁺ celler¹⁵^,¹⁹^,²⁰^,²¹. Derfor, isolation af OLs på P7 kunne sikre høj cellulære udbytte.

I den sidste og fjerde fase af OL udvikling express modne OLs myelinating proteiner (myelin grundlæggende protein (MBPS), proteolipid protein (PLP), myelin forbundet glycoprotein (MAG) og myelin oligodendrocyte glycoprotein (MOG)²²^,²³ ^,²⁴^,²⁵^,²⁶. På dette stadium, modne OLs udvide membraner denne form kompakt enwrapping skafter omkring axoner og er i stand til at myelinate. Dette falder sammen med den iagttagelse, at i rotter og mus hjerne MBP⁺ celler bliver mere og mere rigelige på P14¹⁹^,²⁰^,²¹.

Siden den første dyrkning af oligodendrocyte af Fewster og kolleger i 1967²⁷, er blevet gennemført flere metoder til isolering af OLs fra gnaver CNS herunder immunopanning²⁸^,²⁹^,³⁰, Fluorescens-aktiveret celle sortering (FACS) at udnytte celle overflade-specifikke antigener²⁸^,³¹, differential gradient centrifugering³²^,³³^,³⁴^,³⁵og en rystende metode baseret på differentierede overholdelse af forskellige CNS glia³⁶^,³⁷. Eksisterende kultur metoder har imidlertid begrænsninger, navnlig med hensyn til renhed, udbytte og tid, der kræves til at udføre procedurer³⁸. Derfor er mere effektiv isolation metoder for oligodendrocytes påkrævet.

I dette papir, præsenterer vi en enkel og effektiv udvælgelsesmetode til immunomagnetic isolering af fase tre O4⁺ preoligodendrocytes celler fra neonatal mus unger. Denne metode er en ændring af de teknikker, der er rapporteret af smergel et al. ³⁹ og Dincman et al. ⁴⁰ og giver en oligodendrocyte forberedelse renhed over 80% i ca 4 h.

Protocol

Musene brugt i denne undersøgelse blev passet efter retningslinjerne i SUNY Downstate Medical Center Division af Laboratory Animal ressourcer (DLAR) protokolnummer 15-10492. Bemærk: Primære oligodendrocytes blev isoleret fra neonatal (P5-P7 vildtype C57Bl/6N) mus. På dette stadium udgør umodne OLs mere end 80% af den gnavere hvide substans at sikre høj cellulære udbytte. Alle buffer og reagens kompositioner er tilgængelige for enden af Bordet af materialer. <p clas…

Representative Results

Formålet med denne undersøgelse var at etablere en bedre isolering metode for O4+ primære mus oligodendrocytes kræver mindst mulig manipulation af target-cellerne. Hele proceduren fra eutanasi af hvalpene til plating celler i coverslips tager omkring 4 h og data, der vises her repræsenterer tre uafhængige forsøg. Efter væv dissociation, et gennemsnit på 4,3 ± 0,46 x 107 celler blev isoleret for hvert uafhængigt eksperiment med en levedygtighed af 91% ± 5,…

Discussion

I denne meddelelse præsenterer vi en metode til effektiv isolering af højtrenset umodne mus oligodendrocyte kulturer. I forhold til tidligere publicerede protokoller³⁹^,⁴⁰, gav denne metode et højere renhed med et meget lavere niveau af GFAP-positive astrocytter og en meget lille procentdel af andre celler, ikke-præget. Det er vigtigt at påpege, at disse er umodne OLs allerede forpligtet til oligodendroglial slægt. Disse celler vil således ikke være nyttig…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Denne undersøgelse blev støttet af tilskud fra den National dissemineret sklerose samfundet (RG4591A1/2) og National Institutes of Health (R03NS06740402). Forfatterne takke Dr. Ivan Hernandez og hans lab medlemmer for laboratorium plads, udstyr og rådgivning.

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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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).