Udarbejdelse af Rat oligodendrocyt Stamfader Cultures og Kvantificering af Oligodendrogenesis Brug Dual-infrarød Fluorescens Scanning
Summary
Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.
Abstract
Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.
Introduction
Effektiv nerveledningen i pattedyrs centralnervesystem (CNS) kræver myelinering af axoner ved oligodendrocytter. Under udvikling, opstår oligodendrocytter fra en pulje af oligodendrocyt progenitorceller (OPCs), der menes at migrere fra de ventrikulære zoner af det udviklende forhjernen og neuralrøret 1. Efter migration OPCs differentiere til modne, myelinerende oligodendrocytter, der ikke kun lette effektiv impuls formering, men også give axoner med trofisk støtte 2. Den voksne CNS opretholder en rigelig bestand af OPCs der er spredt over hele det grå og hvid substans omfattende ca. 5-8% af alle celler 3. Efter en demyeliniserende fornærmelse, OPCs migrere til stedet for skaden, formere, og differentierer at erstatte mistede eller beskadigede myelinskeder om eksponering axoner. Men i nogle sygdom / skade indstillinger, er denne proces sig at være ineffektive eller kan svigte helt 4. Menskronisk demyelinisering menes at føje til sygdomsbyrden, effektiv oligodendrogenesis og remyelinering kan lindre symptomerne 5. Det har derfor været af interesse at undersøge OPCs in vitro for at bestemme virkningen af eksperimentelle faktorer på oligodendrogenesis.
Indsigt i de forskellige faser af oligodendrocyt differentiering er blevet muliggjort ved identifikation af fase specifikke cellemarkører. Selvfornyende tidlige progenitorceller er defineret ved deres ekspression af blodpladeafledt vækstfaktor-receptor alpha (PDGFRα), neural / glia antigen 2 (NG2) og A2B5 6 – 8. Som OPCs indleder deres differentiering program og trække sig ud af cellecyklus, de nedregulerer ekspressionen af disse markører og begynder at udtrykke proteiner indikerer premyelinating oligodendrocytter herunder Cyklisk-nukleotid 3'-phosphodiesterase (CNPase) og O4. Endelig deres differentiering til mere modne oligodendrocytes er kendetegnet ved ekspressionen af myelin-associerede proteiner, herunder myelin-associeret glycoprotein (MAG), proteolipidprotein (PLP), og myelin basisk protein (MBP). MBP er et intracellulært perifer membranprotein og en hovedbestanddel af myelinskeden. Mus mangler MBP udvikle en alvorlig fænotype, hvor CNS myelinering markant er faldet fører til rystelser, kramper og tidlig død 9,10. Denne vigtige rolle af MBP i myelinering har ført til dens anvendelse som en markør for oligodendrocyt differentiering både in vitro og in vivo 11.
Kvantificering af MBP kan opnås med flere forskellige metoder. RT-PCR og Western blot-analyse muliggør kvantificering af MBP niveauer under forskellige behandlingsregimer. Immuncytokemi er en fælles kvalitativ tilgang, der kan også være kvantitativ, når der anvendes kamera-baserede mikroskopi tilgange. Selv om disse systemer er pålidelige og grundlæggende forstudiet af oligodendrocyt differentiering, de hver især har deres egne ulemper, der begrænser deres anvendelse i screening narkotika. Først, mængden af primære OPCs at der er behov for RT-PCR og Western blot-analyse reducerer antallet af variabler, der kan undersøges samtidig. Mens kravet cellen for immuncytokemi er meget lavere, skal betydelig tid afsættes til hvert forsøg, hvis kvantificering er målet. Talrige billeder skal opfanges og kvantificeres dernæst at lette eksperimentel analyse. Disse forbehold bliver vigtige hindringer overveje for undersøgelser, der kræver high throughput vurdering. Her beskriver vi en fremgangsmåde, der udnytter grundlæggende aspekter fra både immunocytokemi og Western blot metoder til myelin kvantificering, mens en markant nedsættelse både antallet af celler, der kræves og tid til at fuldføre kvantitativ analyse.
Protocol
Representative Results
Discussion
Protokollen præsenteres her beskriver en hurtig og pålidelig fremgangsmåde til isolering rotte OPCs og kvantificere in vitro oligodendrogenesis som reaktion på eksperimentelle faktorer. Ved hjælp af positiv selektion, er høje udbytter af levedygtige og rene OPCs anvendes direkte til forsøg. Denne metode strømliner isolation, dyrkning og differentieringsfaktorer skridt ind en 1 uge tidsramme, og fikserede celler kan hensigtsmæssigt opbevares ved 4 ° C i adskillige uger uden tab i analysesensitivitet. <…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Grant sponsor: National Institutes of Health; Grant nummer: R37 NS041435.
Materials
| Dissection Materials | |||
| PBS without Ca2+ and Mg2+ | Mediatech | 21-040-CV | 1 x |
| 10 cm Polystyrene Petri Dishes | Fisherbrand | 0857512 | |
| Light Dissection Microscope | Motic | SM2-168 | |
| Dissociation Materials | |||
| Tube Rotator | Miltenyi Biotec | 130-090-753 | Dissociation Tube Rotator |
| Neural Tissue Dissociation Kit (P) | Miltenyi Biotec | 130-092-628 | Enzymatic Dissociation Kit |
| PBS with Ca2+ and Mg2+ | Corning | 20-030-CV | 1 x |
| 40 μm Cell Strainer | Corning | 352340 | |
| A2B5 Column Purification | |||
| Anti-A2B5 Microbeads | Miltenyi Biotec | 130-097-864 | Bead Bound A2B5 Antibody |
| LS Columns | Miltenyi Biotec | 130-042-401 | Magnetic Selection Columns |
| EDTA | Corning | 46-034-Cl | 2mM |
| PBS with Ca2+ and Mg2+ | Corning | 20-030-CV | 1 x |
| Bovine Serum Albumin | Sigma-Aldrich | A9647 | 0.50% |
| Culture Materials | |||
| PDGF-AA | PeproTech Inc | 100-13A | 20 ng/mL |
| Dimethyl sulfoxide | Sigma-Aldrich | D2650 | Hybri-Max 100 mL |
| Triiodothyronine | Sigma-Aldrich | T6397 | 45 nM |
| Clemastine fumarate salt | Sigma-Aldrich | SML0445-100MG | 1 μM |
| Quetiapine hemifumarate salt | Sigma-Aldrich | Q3638-10MG | 1 μM |
| N-acetyl cysteine | Sigma-Aldrich | A9165 | 5 μg/mL |
| Progesterone | Sigma-Aldrich | P8783 | 60 ng/mL |
| Poly-L-lysine | Sigma-Aldrich | P1524 | 10 μg/mL |
| Putrecine | Sigma-Aldrich | P7505 | 16 μg/mL |
| Sodium Selenite | Sigma-Aldrich | S5261 | 40 ng/mL |
| Hydrocortisone | Sigma-Aldrich | H0135 | 50 ng/mL |
| d-Biotin | Sigma-Aldrich | B4639 | 10 ng/mL |
| Insulin | Sigma-Aldrich | I6634 | 5 μg/mL |
| Apo-Transferrin | Sigma-Aldrich | T1147 | 100 μg/mL |
| Bovine Serum Albumin | Sigma-Aldrich | A4161 | 100 μg/mL |
| Trace Elements B | Corning | 25-022-Cl | 1 x |
| B-27 Supplement | Life Technologies | 17504044 | 1 x |
| Penicillin/Streptomycin | Life Technologies | 15140122 | 1 x |
| DMEM | Life Technologies | 11995-065 | 1 x |
| 24-well Black Visiplate with lid | Perkin Elmer | 1450-605 | Tissue culture grade |
| Immunocytochemistry and Quantification | |||
| PFA | Sigma-Aldrich | 100-13A | 4% |
| Triton X-100 | Sigma-Aldrich | 78787 | 0.10% |
| Normal Goat Serum | Jackson Immuno Research | 005-000-121 | 5% |
| mouse monoclonal anti-MBP | Biolegend | SMI-99P | 1:1000 Dilution |
| rabbit polyclonal anti-Actin | Santa Cruz Biotecnology | SC-7210 | 1:200 Dilution |
| rabbit polyclonal anti-Olig2 | Millipore | AB9610 | 1:1000 Dilution |
| goat anti-mouse 680RD | Licor | 926-68070 | 1:500 Dilution |
| goat anti-rabbit 800CW | Licor | 926-32211 | 1:500 Dilution |
| Alexa Fluor 594 goat anti-mouse | Life Technologies | A11032 | 1:000 dilution |
| Alexa Fluor 488 goat anti-rabbit | Life Technologies | A11008 | 1:000 dilution |
| Prolong Gold antifade with DAPI | Life Technologies | P36931 | |
| DPBS with Ca2+ and Mg2+ | Corning | 21-030-CV | |
| Licor Odyssey Clx Infared Imaging System | Licor | ||
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