Summary

Rapido e specifico immunomagnetica isolamento degli oligodendrociti primario del Mouse

Published: May 21, 2018
doi:

Summary

Descriviamo l’isolamento immunomagnetica degli oligodendrociti primario del mouse, che permette l’isolamento rapido e specifico delle cellule per coltura in vitro .

Abstract

L’efficiente e robusto isolamento e coltura dei oligodendrocytes primario (OLs) è uno strumento prezioso per lo Studio in vitro di sviluppo di oligodendroglia, nonché la biologia di demyelinating malattie come la sclerosi multipla e Malattia di Pelizaeus-Merzbacher-simile (PMLD). Qui, presentiamo un semplice e metodo di selezione efficace per l’isolamento di immunomagnetica di fase tre O4+ cellule preoligodendrocytes da cuccioli di topi neonatali. Poiché immaturo OL costituiscono più dell’80% della materia bianca roditore-cervello postnatale giorno 7 (P7) questo metodo di isolamento non solo assicura un alto rendimento cellulare, ma anche l’isolamento specifico di OLs già commesso alla stirpe oligodendroglial, diminuendo il possibilità di isolare cellule contaminanti quali gli astrociti e altre cellule del cervello del mouse. Questo metodo è una modifica delle tecniche segnalato precedentemente e fornisce del oligodendrocyte preparazione purezza superiore al 80% in circa 4 h.

Introduction

Oligodendrociti (OLs) sono le cellule di mielinizzazione del sistema nervoso centrale (SNC)1. L’isolamento e la coltura degli oligodendrociti primari in un ambiente strettamente regolamentato è uno strumento prezioso per lo Studio in vitro di sviluppo di oligodendroglia, nonché la biologia di demyelinating malattie come la sclerosi multipla2 . Ciò richiede un’efficiente e robusto del oligodendrocyte isolamento e coltura metodo3. In questo studio, abbiamo approfittato dell’espressione di un indicatore della superficie delle cellule del oligodendrocyte distintivo per implementare una tecnica di isolamento modificate che è rapido e specifico.

Sono state identificate quattro distinte fasi di maturazione del oligodendrocyte, ognuna caratterizzata dall’espressione di marcatori di superficie cellulare distintivo per ogni stadio di sviluppo (Figura 1). Questi indicatori di superficie delle cellule possono essere riconosciuti da anticorpi specifici4,5e possono essere utilizzati per isolare OLs alle fasi specifiche. Nella prima fase, le cellule del precursore del oligodendrocyte (OPCs) hanno la capacità di proliferare, migrare e rapidi in particolare fattore di crescita derivato dalle piastrine (PDGF-Rα) recettore6, ganglioside A2B5, proteoglicano NG27,8 , neuro-acido polisialico delle cellule adesione molecola9 e grassi-acido-legante proteina 7 (FABP7)10. Gli OPC hanno morfologia bipolare con alcuni processi brevi che emana dai poli opposti del corpo cellulare, che è caratteristico di cellule precursori neurali11.

Figure 1
Figura 1: espressione di marcatori di superficie delle cellule durante lo sviluppo del oligodendrocyte mouse. OLs cella marcatori di superficie come A2B5, GalC (O1), NG2, O4 e PDGF-Rα può essere utilizzato per isolare specificamente oligodendrociti in fase inerente allo sviluppo specifica utilizzando anticorpi specifici.   Clicca qui per visualizzare una versione più grande di questa figura.

Nella seconda fase, OPCs danno luogo a preoligodendrocytes e rapidi alla membrana delle cellule non solo gli indicatori OPC, ma anche il sulfatide (un galattolipide solfonata) riconosciuto dal O4 anticorpo12,13e la proteina GPR1714, che persiste fino alla fase di oligodendrociti immaturi (OL). In questa fase, preoligodendrocytes estendere multipolari processi brevi. Preoligodendrocytes sono la grande fase OL al giorno postnatale 2 (P2) nella materia bianca cerebrale di ratto e topo con una popolazione secondaria di immaturi OLs15.

Durante la terza tappa, immaturi OLs continuano a esprimere O4, perdere espressione degli indicatori A2B5 e NG2 e cominciano ad esprimere Galattocerebrosidi C16. In questa fase, OLs sono impegnati alla stirpe oligodendroglial e diventare cellule post-mitotiche con rami lunghi ramificate17,18. Immaturo OL costituiscono più dell’80% della materia bianca del roditore in P7 e in questo momento si osservano le prime cellule MBP+ 15,19,20,21. Di conseguenza, isolamento di OLs presso P7 potrebbe garantire alto rendimento cellulare.

Nella fase finale e la quarta di sviluppo OL, OLs matura esprimono proteine myelinating (proteina basica della mielina (MBP), proteolipide (PLP), glicoproteina mielina associata (MAG) e myelin oligodendrocyte glycoprotein (MOG)22,23 ,24,25,26. In questa fase, OLs matura estendere membrane quello compatto di forma economale guaine intorno gli assoni e sono in grado di mielinare. Questo coincide con l’osservazione che nel cervello di ratto e topo, cellule MBP+ diventano sempre più abbondanti a P1419,20,21.

Poiché il primo isolamento del oligodendrocyte Fewster e colleghi nel 196727, sono stati implementati diversi metodi per l’isolamento di OLs da roditore SNC compreso immunopanning28,29,30, fluorescenza-attivato delle cellule ordinano (FACS) sfruttando delle cellule degli antigeni di superficie-specific28,31, differenziale mediante centrifugazione in gradiente32,33,34,35 e agitazione metodo basato sull’adesione differenziale di diverse CNS glia36,37. Tuttavia, i metodi di coltura esistenti hanno limitazioni, soprattutto in termini di purezza, rendimento e tempo necessario per eseguire la procedure38. Pertanto, i metodi più efficienti di isolamento per gli oligodendrociti sono necessari.

In questa carta, presentiamo un semplice e metodo di selezione efficace per l’isolamento di immunomagnetica di fase tre O4+ cellule preoligodendrocytes da cuccioli di topi neonatali. Questo metodo è una modificazione delle tecniche riportate da Emery et al. 39 e Dincman et al. 40 e fornisce una purezza di preparazione del oligodendrocyte superiore all’80% in circa 4 h.

Protocol

I topi utilizzati in questo studio sono stati curati per secondo le indicazioni del numero di protocollo di SUNY Downstate Medical Center divisione del laboratorio animale risorse (DLAR) 15-10492. Nota: Oligodendrocytes primario sono stati isolati da neonatale (P5-P7 selvaggio-tipo C57Bl/6N) topi. In questa fase, immaturi OLs costituiscono più dell’80% della materia bianca del roditore garantire alto rendimento cellulare. Tutti i buffer e composizioni di reagente sono disponibili alla fine de…

Representative Results

Lo scopo di questo studio era di stabilire un metodo di isolamento migliorato per O4+ oligodendrociti primario del mouse che richiedono la manipolazione meno possibile delle cellule bersaglio. L’intera procedura dall’eutanasia dei cuccioli di placcatura delle celle di vetrini coprioggetti prende circa 4 h e i dati qui indicati rappresentano tre esperimenti indipendenti. Dopo dissociazione del tessuto, una media di 4,3 ± 0,46 x 107 cellule sono state isolate per ogni…

Discussion

In questa comunicazione, presentiamo un metodo per l’isolamento efficiente delle culture del oligodendrocyte altamente purificata del mouse immaturo. Rispetto ai protocolli precedentemente pubblicati39,40, questo metodo ha reso una purezza più elevata con un livello molto più basso degli astrociti GFAP-positive e una percentuale molto bassa di altre cellule non caratterizzato. È importante sottolineare che questi sono immaturi OLs già commit alla stirpe oligo…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Questo studio è stato sostenuto da sovvenzioni dal National Multiple Sclerosis Society (RG4591A1/2) e il National Institutes of Health (R03NS06740402). Gli autori ringraziano il Dr. Ivan Hernandez e membri del suo laboratorio per fornire consulenza, attrezzature e lo spazio laboratorio.

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))

References

  1. Emery, B. Regulation of oligodendrocyte differentiation and myelination. Science. 330 (6005), 779-782 (2010).
  2. Yang, Z., Watanabe, M., Nishiyama, A. Optimization of oligodendrocyte progenitor cell culture method for enhanced survival. J Neurosci Methods. 149 (1), 50-56 (2005).
  3. Niu, J., et al. An efficient and economical culture approach for the enrichment of purified oligodendrocyte progenitor cells. J Neurosci Methods. 209 (1), 241-249 (2012).
  4. Zhang, S. C. Defining glial cells during CNS development. Nat Rev Neurosci. 2 (11), 840-843 (2001).
  5. Pfeiffer, S. E., Warrington, A. E., Bansal, R. The oligodendrocyte and its many cellular processes. Trends Cell Biol. 3 (6), 191-197 (1993).
  6. Hart, I. K., Richardson, W. D., Heldin, C. H., Westermark, B., Raff, M. C. PDGF receptors on cells of the oligodendrocyte-type-2 astrocyte (O-2A) cell lineage. Development. 105 (3), 595-603 (1989).
  7. Nishiyama, A., Lin, X. H., Giese, N., Heldin, C. H., Stallcup, W. B. Interaction between NG2 proteoglycan and PDGF alpha-receptor on O2A progenitor cells is required for optimal response to PDGF. J Neurosci Res. 43 (3), 315-330 (1996).
  8. Pringle, N. P., Mudhar, H. S., Collarini, E. J., Richardson, W. D. PDGF receptors in the rat CNS: during late neurogenesis, PDGF alpha-receptor expression appears to be restricted to glial cells of the oligodendrocyte lineage. Development. 115 (2), 535-551 (1992).
  9. Grinspan, J. B., Franceschini, B. Platelet-derived growth factor is a survival factor for PSA-NCAM+ oligodendrocyte pre-progenitor cells. J Neurosci Res. 41 (4), 540-551 (1995).
  10. Sharifi, K., et al. Differential expression and regulatory roles of FABP5 and FABP7 in oligodendrocyte lineage cells. Cell Tissue Res. 354 (3), 683-695 (2013).
  11. Chittajallu, R., Aguirre, A., Gallo, V. NG2-positive cells in the mouse white and grey matter display distinct physiological properties. J Physiol. 561 (Pt 1), 109-122 (2004).
  12. Bansal, R., Warrington, A. E., Gard, A. L., Ranscht, B., Pfeiffer, S. E. Multiple and novel specificities of monoclonal antibodies O1, O4, and R-mAb used in the analysis of oligodendrocyte development. J Neurosci Res. 24 (4), 548-557 (1989).
  13. Sommer, I., Schachner, M. Monoclonal antibodies (O1 to O4) to oligodendrocyte cell surfaces: an immunocytological study in the central nervous system. Dev Biol. 83 (2), 311-327 (1981).
  14. Boda, E., et al. The GPR17 receptor in NG2 expressing cells: focus on in vivo cell maturation and participation in acute trauma and chronic damage. Glia. 59 (12), 1958-1973 (2011).
  15. Dean, J. M., et al. Strain-specific differences in perinatal rodent oligodendrocyte lineage progression and its correlation with human. Dev Neurosci. 33 (3-4), 251-260 (2011).
  16. Yu, W. P., Collarini, E. J., Pringle, N. P., Richardson, W. D. Embryonic expression of myelin genes: evidence for a focal source of oligodendrocyte precursors in the ventricular zone of the neural tube. Neuron. 12 (6), 1353-1362 (1994).
  17. Armstrong, R. C., Dorn, H. H., Kufta, C. V., Friedman, E., Dubois-Dalcq, M. E. Pre-oligodendrocytes from adult human CNS. J Neurosci. 12 (4), 1538-1547 (1992).
  18. Gard, A. L., Pfeiffer, S. E. Oligodendrocyte progenitors isolated directly from developing telencephalon at a specific phenotypic stage: myelinogenic potential in a defined environment. Development. 106 (1), 119-132 (1989).
  19. Bjelke, B., Seiger, A. Morphological distribution of MBP-like immunoreactivity in the brain during development. Int J Dev Neurosci. 7 (2), 145-164 (1989).
  20. Hardy, R. J., Friedrich, V. L. Progressive remodeling of the oligodendrocyte process arbor during myelinogenesis. Dev Neurosci. 18 (4), 243-254 (1996).
  21. Hartman, B. K., Agrawal, H. C., Kalmbach, S., Shearer, W. T. A comparative study of the immunohistochemical localization of basic protein to myelin and oligodendrocytes in rat and chicken brain. J Comp Neurol. 188 (2), 273-290 (1979).
  22. Wei, Q., Miskimins, W. K., Miskimins, R. Stage-specific expression of myelin basic protein in oligodendrocytes involves Nkx2.2-mediated repression that is relieved by the Sp1 transcription factor. J Biol Chem. 280 (16), 16284-16294 (2005).
  23. Stolt, C. C., et al. Terminal differentiation of myelin-forming oligodendrocytes depends on the transcription factor Sox10. Genes Dev. 16 (2), 165-170 (2002).
  24. Emery, B., et al. Myelin gene regulatory factor is a critical transcriptional regulator required for CNS myelination. Cell. 138 (1), 172-185 (2009).
  25. Reynolds, R., Wilkin, G. P. Development of macroglial cells in rat cerebellum. II. An in situ immunohistochemical study of oligodendroglial lineage from precursor to mature myelinating cell. Development. 102 (2), 409-425 (1988).
  26. Scolding, N. J., et al. Myelin-oligodendrocyte glycoprotein (MOG) is a surface marker of oligodendrocyte maturation. J Neuroimmunol. 22 (3), 169-176 (1989).
  27. Fewster, M. E., Scheibel, A. B., Mead, J. F. The preparation of isolated glial cells from rat and bovine white matter. Brain Res. 6 (3), 401-408 (1967).
  28. Gard, A. L., Williams, W. C., Burrell, M. R. Oligodendroblasts distinguished from O-2A glial progenitors by surface phenotype (O4+GalC-) and response to cytokines using signal transducer LIFR beta. Dev Biol. 167 (2), 596-608 (1995).
  29. Gard, A. L., Pfeiffer, S. E. Glial cell mitogens bFGF and PDGF differentially regulate development of O4+GalC- oligodendrocyte progenitors. Dev Biol. 159 (2), 618-630 (1993).
  30. Barres, B. A., Raff, M. C. Proliferation of oligodendrocyte precursor cells depends on electrical activity in axons. Nature. 361 (6409), 258-260 (1993).
  31. Behar, T., McMorris, F. A., Novotny, E. A., Barker, J. L., Dubois-Dalcq, M. Growth and differentiation properties of O-2A progenitors purified from rat cerebral hemispheres. J Neurosci Res. 21 (2-4), 168-180 (1988).
  32. Vitry, S., Avellana-Adalid, V., Lachapelle, F., Baron-Van Evercooren, A. Migration and multipotentiality of PSA-NCAM+ neural precursors transplanted in the developing brain. Mol Cell Neurosci. 17 (6), 983-1000 (2001).
  33. Duncan, I. D., Paino, C., Archer, D. R., Wood, P. M. Functional capacities of transplanted cell-sorted adult oligodendrocytes. Dev Neurosci. 14 (2), 114-122 (1992).
  34. Goldman, J. E., Geier, S. S., Hirano, M. Differentiation of astrocytes and oligodendrocytes from germinal matrix cells in primary culture. J Neurosci. 6 (1), 52-60 (1986).
  35. Althaus, H. H., Montz, H., Neuhoff, V., Schwartz, P. Isolation and cultivation of mature oligodendroglial cells. Naturwissenschaften. 71 (6), 309-315 (1984).
  36. McCarthy, K. D., de Vellis, J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol. 85 (3), 890-902 (1980).
  37. Szuchet, S., Yim, S. H. Characterization of a subset of oligodendrocytes separated on the basis of selective adherence properties. J Neurosci Res. 11 (2), 131-144 (1984).
  38. Chew, L. J., DeBoy, C. A., Senatorov, V. V. Finding degrees of separation: experimental approaches for astroglial and oligodendroglial cell isolation and genetic targeting. J Neurosci Methods. 236, 125-147 (2014).
  39. Emery, B., Dugas, J. C. Purification of oligodendrocyte lineage cells from mouse cortices by immunopanning. Cold Spring Harb Protoc. 2013 (9), 854-868 (2013).
  40. 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 (2012).
  41. Buttery, P. C., ffrench-Constant, C. Laminin-2/integrin interactions enhance myelin membrane formation by oligodendrocytes. Mol Cell Neurosci. 14 (3), 199-212 (1999).
  42. Chun, S. J., Rasband, M. N., Sidman, R. L., Habib, A. A., Vartanian, T. Integrin-linked kinase is required for laminin-2-induced oligodendrocyte cell spreading and CNS myelination. J Cell Biol. 163 (2), 397-408 (2003).
  43. Colognato, H., Ramachandrappa, S., Olsen, I. M., ffrench-Constant, C. Integrins direct Src family kinases to regulate distinct phases of oligodendrocyte development. J Cell Biol. 167 (2), 365-375 (2004).
  44. ffrench-Constant, C., Colognato, H. Integrins: versatile integrators of extracellular signals. Trends Cell Biol. 14 (12), 678-686 (2004).
  45. Oh, L. Y., Yong, V. W. Astrocytes promote process outgrowth by adult human oligodendrocytes in vitro through interaction between bFGF and astrocyte extracellular matrix. Glia. 17 (3), 237-253 (1996).
  46. Besnard, F., Perraud, F., Sensenbrenner, M., Labourdette, G. Effects of acidic and basic fibroblast growth factors on proliferation and maturation of cultured rat oligodendrocytes. Int J Dev Neurosci. 7 (4), 401-409 (1989).
  47. Armstrong, R., Friedrich, V. L., Holmes, K. V., Dubois-Dalcq, M. In vitro analysis of the oligodendrocyte lineage in mice during demyelination and remyelination. J Cell Biol. 111 (3), 1183-1195 (1990).
  48. Grinspan, J. B., Stern, J. L., Franceschini, B., Pleasure, D. Trophic effects of basic fibroblast growth factor (bFGF) on differentiated oligodendroglia: a mechanism for regeneration of the oligodendroglial lineage. J Neurosci Res. 36 (6), 672-680 (1993).
  49. Mason, J. L., Goldman, J. E. A2B5+ and O4+ Cycling progenitors in the adult forebrain white matter respond differentially to PDGF-AA, FGF-2, and IGF-1. Mol Cell Neurosci. 20 (1), 30-42 (2002).
  50. Schildge, S., Bohrer, C., Beck, K., Schachtrup, C. Isolation and culture of mouse cortical astrocytes. J Vis Exp. (71), (2013).

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Cite This Article
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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