Name: rapid and specific immunomagnetic isolation of mouse primary oligodendrocytes
Uploaded: 2018-05-21T14:30:00
Description: Watch this Scientific Journal Video about Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes at JoVE.com

This study was supported by grants from the National Multiple Sclerosis Society (RG4591A1/2) and the National Institutes of Health (R03NS06740402). The authors thank Dr. Ivan Hernandez and his lab members for providing laboratory space, equipment and advice.

The mice used in this study were cared for according to the guidelines of the SUNY Downstate Medical Center Division of Laboratory Animal Resources (DLAR) protocol number 15-10492.
NOTE: Primary oligodendrocytes were isolated from neonatal (P5-P7 wild-type C57Bl/6N) mice. At this stage, immature OLs constitute more than 80% of the rodent white matter ensuring high cellular yield. All buffer and reagent compositions are available at the end of the Table of Materials.
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In this communication, we present a method for the efficient isolation of highly purified immature mouse oligodendrocyte cultures. Compared to previously published protocols39,40, this method yielded a higher purity with a much lower level of GFAP-positive astrocytes and a very low percentage of other non-characterized cells. It is important to point out that these are immature OLs already committed to the oligodendroglial lineage. Thus, these cells would not be ...

Oligodendrocytes (OLs) are the myelinating cells of the central nervous system (CNS)1. The isolation and culture of primary oligodendrocytes in a tightly regulated environment is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis2. This requires an efficient and robust oligodendrocyte isolation and culture method3. In this study, we took advantage of the expression of a distinctive oligodendrocyte cell surface marker to implement a modified isolation technique that is rapid and specific.
Four distinct stages of oligodendrocyte maturation have been identified, each characterized by the expression of distinctive cell surface markers for each developmental stage (Figure 1). These cell surface markers can be recognized by specific antibodies4,5, and can be used to isolate OLs at specific stages. In the first stage, oligodendrocyte precursor cells (OPCs) have the capacity to proliferate, migrate, and specifically express platelet-derived growth factor receptor (PDGF-R&#945;)6, ganglioside A2B5, proteoglycan NG27,8, polysialic acid-neural cell adhesion molecule9 and fatty-acid-binding protein 7 (FABP7)10. OPCs have bipolar morphology with few short processes emanating from the opposing poles of the cell body, which is characteristic of neural precursor cells11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57543/57543fig1v2.jpg" /> 
Figure 1: Expression of cell surface markers during the mouse oligodendrocyte development.&#160;OLs cell surface markers such as A2B5, GalC (O1), NG2, O4, and PDGF-R&#945; can be used to specifically isolate oligodendrocytes at specific developmental stage by using specific antibodies.&#160;<a href="//cloudflare.jove.com/files/ftp_upload/57543/57543fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In the second stage, OPCs give rise to preoligodendrocytes and express at the cell membrane not only OPC markers, but also the sulfatide (a sulfated galactolipid) recognized by the O4 antibody12,13, and the GPR17 protein14, which persists until the immature oligodendrocyte (OL) stage. At this stage, preoligodendrocytes extend multipolar short processes. Preoligodendrocytes are the major OL stage at postnatal day 2 (P2) in the cerebral white matter of both rat and mouse with a minor population of immature OLs15.
During the third stage, immature OLs continue to express O4, lose expression of A2B5 and NG2 markers and begin to express galactocerebroside C16. At this stage, OLs are committed to the oligodendroglial lineage and become post-mitotic cells with long ramified branches17,18. Immature OL constitute more than 80% of the rodent white matter at P7 and at this time the first MBP+ cells are observed15,19,20,21. Therefore, isolation of OLs at P7 could ensure high cellular yield.
In the final and fourth stage of OL development, mature OLs express myelinating proteins (myelin basic protein (MBP), proteolipid protein (PLP), myelin associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG)22,23,24,25,26. At this stage, mature OLs extend membranes that form compact enwrapping sheaths around the axons and are able to myelinate. This coincides with the observation that in rat and mouse brain, MBP+ cells become increasingly abundant at P1419,20,21.
Since the first isolation of oligodendrocyte by Fewster and colleagues in 196727, several methods for isolation of OLs from the rodent CNS have been implemented including immunopanning28,29,30, fluorescence-activated cell sorting (FACS) exploiting cell surface-specific antigens28,31, differential gradient centrifugation32,33,34,35 and a shaking method based on differential adherence of different CNS glia36,37. However, existing culture methods have limitations, particularly in terms of purity, yield and time required to perform the procedures38. Therefore, more efficient isolation methods for oligodendrocytes are required.
In this paper, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. This method is a modification of the techniques reported by Emery et al.39 and Dincman et al.40 and provides an oligodendrocyte preparation purity above 80% in about 4 h.

<table>
	<tbody>
		<tr>
			<td>10ml serological pipets</td>
			<td>Fisher Scientific</td>
			<td>13-676-10J</td>
			<td></td>
		</tr>
		<tr>
			<td>10ml syringe Luer-Loc tip</td>
			<td>BD, Becton Dickinson</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml conical tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture plates</td>
			<td>Falcon</td>
			<td>353935</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#181;m cell strainer</td>
			<td>Fisher Scientific</td>
			<td>22368547</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml conical tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml serological pipets</td>
			<td>Fisher Scientific</td>
			<td>13-676-10H</td>
			<td></td>
		</tr>
		<tr>
			<td>60mm tissue culture plates</td>
			<td>Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>70&#181;m cell strainer</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse IgG (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-rabbit IgM (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A21042</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-rabbit IgM (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 goat anti-chicken IgG (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11042</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-O4 beads- Anti-O4MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-543</td>
			<td></td>
		</tr>
		<tr>
			<td>Apo-Transferrin human</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Autofil complete bottle top filter assembly, 0.22um filter, 250ml</td>
			<td>USA Scientific</td>
			<td>6032-1101</td>
			<td></td>
		</tr>
		<tr>
			<td>Autofil complete bottle top filter assembly, 0.22um filter, 250ml</td>
			<td>USA Scientific</td>
			<td>6032-1102</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma</td>
			<td>B7660</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Growth Serum (BGS)</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30541.03</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher Scientific</td>
			<td>BP-1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>CNTF</td>
			<td>Peprotech</td>
			<td>450-50</td>
			<td></td>
		</tr>
		<tr>
			<td>d-Biotin</td>
			<td>Sigma</td>
			<td>B4639</td>
			<td></td>
		</tr>
		<tr>
			<td>Desoxyribonuclease I (DNAse I)</td>
			<td>Worthington</td>
			<td>LS002007</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>S311</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence microscope with an Olympus DP70 camera</td>
			<td>Olympus</td>
			<td>Bx51</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather disposable scalpels</td>
			<td>Andwin Scientific</td>
			<td>EF7281C</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma</td>
			<td>F6886</td>
			<td></td>
		</tr>
		<tr>
			<td>German glass coverslips, #1 thickness, 12mm diameter round</td>
			<td>NeuVitro</td>
			<td>GG-12-oz</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP antibody</td>
			<td>Aves</td>
			<td>GFAP</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Invitrogen</td>
			<td>35050-61</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Invitrogen</td>
			<td>12585-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic separator stand - MACS multistand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic separator-MiniMACS separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex PES 0.22&#181;m filter unit</td>
			<td>Millipore</td>
			<td>SLG033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media- Prolong Gold with DAPI</td>
			<td>Thermo Fisher</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-cysteine (NAC)</td>
			<td>Sigma</td>
			<td>A8199</td>
			<td></td>
		</tr>
		<tr>
			<td>Natural mouse laminin</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium A</td>
			<td>Invitrogen</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurotrophin-3 (NT-3)</td>
			<td>Peprotech</td>
			<td>450-03</td>
			<td></td>
		</tr>
		<tr>
			<td>NG2 antibody</td>
			<td>Millipore</td>
			<td>AB5320</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>LS003126</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS without Ca2+ and Mg2+</td>
			<td>Sigma</td>
			<td>D5652</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF</td>
			<td>Peprotech</td>
			<td>100-13A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>Invivogen</td>
			<td>ant-pm-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>Selection column-LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Trace elements B</td>
			<td>Corning</td>
			<td>25-000-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Triiodothyronine (T3)</td>
			<td>Sigma</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Corning</td>
			<td>25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>B27NBMA</td>
			<td></td>
			<td></td>
			<td>487.75 mL Neurobasal Medium A; 10 mL B27 Supplement; 1 mL Primocin; 1.25 mL Glutamax; Filter sterilize and store at 4 &#176;C until use.</td>
		</tr>
		<tr>
			<td>B27NBMA + 10% BGS</td>
			<td></td>
			<td></td>
			<td>27 mL B27NBMA; 3 mL Bovine growth serum</td>
		</tr>
		<tr>
			<td>CNTF solution stock (10 &#181;g/ml; 1000X)</td>
			<td></td>
			<td></td>
			<td>Order from Peprotech (450-50). Make up at 0.1 to 1 mg/ml according to Manufacturer&#8217;s instruction (may vary from lot to lot) in buffer (e.g. DPBS + 0.2% BSA). Store at -80 &#176;C. 
			Working solution (10 &#181;g/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 10&#181;g/ml in sterile, chilled 0.2% BSA/DPBS. 
			3. Aliquot (20&#181;l/tube) and snap freeze in liquid nitrogen. 
			4. Store aliquots at -80 &#176;C.</td>
		</tr>
		<tr>
			<td>d-Biotin stock solution (50 &#181;g/ml; 5000X)</td>
			<td></td>
			<td></td>
			<td>Resuspend d-Biotin (Sigma-B4639) in double-distilled H2O at 50 &#181;g/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&#176;C. If the d-Biotin still will not solubilize, it is fine to make up a less concentrated (e.g. 10&#181;g/ml), and to add a higher volume to the B27NBMA (1/1000), instead of 1/5000). Store at 4&#176;C.</td>
		</tr>
		<tr>
			<td>DNase I stock solution</td>
			<td></td>
			<td></td>
			<td>1. Dissolve at 12,500 U Deoxyribonuclease I / ml in HBSS chilled on ice. 
			2. Filter sterilize on ice 
			3. Aliquot at 200 &#181;l and freeze overnight at -20&#176;C. 
			4. Store aliquots at -20 to -30&#176;C.</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (w/o Ca2+ and Mg2+)</td>
			<td></td>
			<td></td>
			<td>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 &#176;C.</td>
		</tr>
		<tr>
			<td>Forskolin stock solution (4.2 mg/ml; 1000X)</td>
			<td></td>
			<td></td>
			<td>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 &#181;l) and store at -20&#176;C.</td>
		</tr>
		<tr>
			<td>Hank&#8217;s balanced salts (HBSS) (Sigma</td>
			<td></td>
			<td></td>
			<td>1. Measure 900 ml of water (temperature 15-20 &#176;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 &#176;C.</td>
		</tr>
		<tr>
			<td>Insulin stock solution (4000 &#181;g/ml)</td>
			<td></td>
			<td></td>
			<td>Thaw the bottle and aliquot 25 &#181;l per microcentrifuge tube and store at -20&#176;C.</td>
		</tr>
		<tr>
			<td>Laminin solution</td>
			<td></td>
			<td></td>
			<td>Slowly thaw laminin in the cold (2&#176;C to 8&#176;C) to avoid gel formation. Then, aliquot into polypropylene tubes. Store at 5&#176; C to -20&#176; C in aliquots (e.g. 20 &#181;l) and do not freeze/thaw repeatedly. Laminin may be stored at these temperatures for up to six months.</td>
		</tr>
		<tr>
			<td>Magnetic Cell Sorting (MCS) Buffer</td>
			<td></td>
			<td></td>
			<td>Prepare the solution containing phosphate-buffered saline (PBS), pH 7.2, and 0.5% bovine serum albumin (BSA), 0.5 mM EDTA, 5&#181;g/ml Insulin, 1 g/L Glucose. Sterilize and degas by filtration the buffer by passing it through a 0.22 &#181;m Millex filter. Store the buffer at 4&#176;C until use</td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine (NAC) stock solution (5mg/ml; 1000X)</td>
			<td></td>
			<td></td>
			<td>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 &#181;l). Store at -20&#176;C.</td>
		</tr>
		<tr>
			<td>NT3 stock solution (1 &#181;g/ml; 1000X)</td>
			<td></td>
			<td></td>
			<td>Master stock: 
			Order from Peprotech (450-03). Make up at 0.1 to 1 mg/ml according to manufacturer&#8217;s instructions (may vary from lot to lot), in buffer (e.g. DPBS + 0.2% BSA). Store at -80&#176;C. 
			 
			Working stock (1&#181;g/ml; 1000X): 
			1. Make on 0.2% BSA in DPBS solution and filter sterilize. 
			2. Dilute master stock aliquot to 1 &#181;g/ml in sterile, chilled 0.2% BSA/DPBS. 
			3. Aliquot (e.g. 20&#181;l/tube) and snap freeze in liquid nitrogen. 
			4. Store aliquots at -80&#176;C.</td>
		</tr>
		<tr>
			<td>PDGF stock solution (10 &#181;g/ml; 1000X)</td>
			<td></td>
			<td></td>
			<td>Master stock: 
			Order from Peprotech (100-13A). Make up at 0.1 to 1 mg/ml according to manufacturer&#8217;s instructions (may vary from lot to lot) in buffer (e.g. DPBS) + 0.2% BSA). Store at -80&#176;C. 
			 
			Working stock (1&#181;g/ml; 1000X): 
			1. Make on 0.2% BSA in DPBS solution and filter sterilize. 
			2. Dilute master stock aliquot to 1&#181;g/ml in sterile, chilled 0.2% BSA/DPBS. 
			3. Aliquot (e.g. 20&#181;l/tube) and snap freeze in liquid nitrogen. 
			4. Store aliquots at -80&#176;C.</td>
		</tr>
		<tr>
			<td>Poly-D-lysine (1mg/ml; 100X)</td>
			<td></td>
			<td></td>
			<td>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. 100&#181;l/tube). Store at -20&#176;C. Prior to use, dilute the 100X stock (1mg/ml) to 50 &#181;g/ml in sterile water.</td>
		</tr>
		<tr>
			<td>Oligodendrocyte proliferation media</td>
			<td></td>
			<td></td>
			<td>see Supplementary Table 1</td>
		</tr>
		<tr>
			<td>Oligodendrocyte differentiation media</td>
			<td></td>
			<td></td>
			<td>see Supplementary Table 1</td>
		</tr>
		<tr>
			<td>Sato supplement (100X)</td>
			<td></td>
			<td></td>
			<td>see Supplementary Table 1</td>
		</tr>
		<tr>
			<td>References: the list of reagents and recipes were adopted from the protocols previously described by Emery et. al. 2013 (Emery, B. &#38; 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. &#38; 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))</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
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rapid and specific immunomagnetic isolation of mouse primary oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

The purpose of this study was to establish an improved isolation method for O4+ primary mouse oligodendrocytes requiring the least possible manipulation of the target cells. The entire procedure from euthanasia of the pups to plating of the cells in coverslips takes about 4 h and data shown here represent three independent experiments. After tissue dissociation, an average of 4.3 &#177; 0.46 x 107 cells were isolated for each independent experiment, with a viability ...

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Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

The overall goal of this technique is to use immunomagnetic isolation to select O4 positive oligodendrocytes from neonatal mice pups for their in vitro culture analysis. This method can help answer key questions in neural science field related to the study of diseases that affect myelin and myelination. The main advantage of this technique is that it facilitates the preparation of final oligodendrocyte culture of a greater than 80%purity in about hour hours.Begin by using small dissecting scissors to cut the skin along the midline of the scalp of a day five to seven postnatal day mouse. Retract the skin flap to expose the skull, and cut the skull carefully along the midline from the opening in the back to the frontal area. From the opening in the back of the skull, cut toward each eye socket along the base of the bone and use fine forceps to gently tease the cortices away from the midbrain.Then, transfer the cortices to a 60 millimeter tissue culture dish containing seven milliliters of B-27 Neurobasal A Medium. When all of the brains have been collected, transfer the cortices into a new 60 millimeter tissue culture dish containing five milliliters of dissociation buffer, and use a number 15 scalpel blade to dice the cortices into one cubed millimeter pieces. Incubate the brain pieces in a 37 degree Celsius 5%CO2 incubator for 20 minutes.Then, add one milliliter of Bovine Growth Serum to stop the enzymatic reaction. Using a 10 milliliter Serological Pipette, transfer the tissue slurry into a 15 milliliter conical tube and gently dissociate the brain tissue six to eight times with pipetting. Allow the dissociated tissue chunks to settle for two to three minutes.Then, transfer the supernatant into a fresh tube. Next, add three milliliters of Neurobasal A Medium, supplemented with Bovine Growth Serum and DNase I to the tissues, and use a five milliliter pipette to gently dissociate the tissues with more pipetting. You see, the appropriate pipetting force is crucial to ensure a high yield of viable cells.If pipetting is too hard, the viability might be low. Whereas if pipetting is too gentle, the cellular yield might be low. Allow the tissue pieces to settle for another two to three minutes.Then, transfer the supernatant into a new tube, and add three milliliters of fresh Neurobasal A Medium, supplemented with Bovine Growth Serum and DNase I to the tissue. Gently dissociate the brain tissue with a P-1000 pipette tip until no large chunk of tissue remain, taking care to avoid bubbles. Use a 10 milliliter pipette to filter the cell solution through a 70 micron cell strainer into a 50 milliliter conical tube, and bring the final volume of the single-cell suspension to 30 milliliters with fresh Neurobasal A Medium, supplemented with Bovine Growth Serum and DNase I.Split the cell suspension to two 15 milliliter conical tubes, and pellet the neural cells by centrifugation.Remove all but the last five to 10 microliters of cloudy supernatant and add three milliliters of Neurobasal A Medium supplemented with Bovine Growth Serum to the cells. Carefully re-suspend the cells pellet and bring the final volume up to 15 milliliters with fresh Neurobasal A Medium containing 10%Bovine Growth Serum. Filter the cell solution through a 40 micron cell strainer into a new 50 milliliter conical tube, and bring the final volume up to 30 milliliters with fresh Neurobasal A Medium containing 10%Bovine Growth Serum.Then, split the filtered cell suspension between two 15 milliliter conical tubes for centrifugation. and re-suspend the pellets in 2.5 milliliters of ice cold Magnetic Cell Sorting Buffer before pooling the cells. After counting, centrifuge the cells again, and re-suspend the pellet in 90 microliters of fresh Magnetic Cell Sorting Buffer per one times 10 to the seven cells.Next, add 10 microliters of anti-04 beads per one times 10 to the seven cells, and mix the cell solution with gentle flicking. After 15 minutes at four degrees Celsius with a gentle flicking every five minutes, gently add two milliliters of Magnetic Cell Sorting Buffer per one times 10 to the seven cells for a wash by centrifugation, and discard the supernatant by careful vacuum aspiration. Re-suspend the pellet in 500 microliters of fresh Magnetic Cell Sorting Buffer for every one times 10 to the seven cells, and place an appropriately sized magnetic beads sorting column into its corresponding magnetic separator.Place a 40 micron strainer onto the column and pre-rinse the strainer with three milliliters of Magnetic Cell Sorting Buffer, letting the buffer run through the column without letting the column dry. Add the cells to the strainer, followed by a one milliliter wash with Magnetic Cell Sorting Buffer. Wash the column three times with three milliliters of Magnetic Cell Sorting Buffer per wash, and one time with Oligodendrocyte Proliferation Medium.Then, transfer the column into a 15 milliliter tube, and use the plunger to immediately flush five milliliters of fresh Oligodendrocyte Proliferation Medium through the column. After counting, dilute the cells to an appropriate plating density, and replace the laminin from pre-coated cover slips in a 24 well plate with 100 microliters of Oligodendrocyte Proliferation Medium. Incubate the oligodendrocytes at 37 degrees Celsius and 5%CO2 for no longer than 45 minutes to promote oligodendrocyte adhesion to the cover slips.Then, flood each well of the 24 well plate with 500 microliters of Oligodendrocyte Proliferation Medium for a 24 hour incubation. The next day, replace the supernatants with Oligodendrocyte Differentiation Medium, and return the cells to the incubator until they are ready for fixation. 24 hours after plating, the cells appear to be bi or tripolar under Phase Contrast Microscopy, a characteristic feature of early stage proliferating oligodendrocyte.Immunofluorescence Staining shows that 24 hours after plating, the majority of these pre-oligodendrocytes also demonstrate NG2 and O4 labeling, while labeling with GFAP and anti-01 antibody are much less common, suggesting that the majority of cells at this stage ae pre-oligodendrocytes with few immature or mature oligodendrocytes. At 72 hours, the oligodendrocyte morphology appears to be more complex than at 24 hours, and there is a much lower frequency of cells positive for the early oligodendrocyte marker, NG2. Also, most cells demonstrate O4 labeling, and almost half of the cells stained for 01 antibody at this stage, suggesting that the cells are differentiating into a more mature phenotype.Notably, the presence of astrocytes remains extremely low. These results indicate that over time, immuno-magnetically isolated oligodendrocytes are able to differentiate in vitro into Stage Three of maturation with very little contamination of other cells types, such as astrocytes. After watching this video, you should have a good understanding of how to isolate primary oligodendrocytes from neonatal mouse pups using immunomagnetic isolation.Once mastered, this technique can be completed in four hours, if it is performed properly. Thanks for watching. Good luck with your experiments.

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