Name: isolation and culture of rodent microglia to promote a dynamic ramified morphology in serum-free medium
Uploaded: 2018-03-09T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium at JoVE.com

Questo lavoro è stato supportato dal Christopher e Dana Reeve Foundation International Research Consortium sulla ferita del midollo spinale, il Dr. Miriam e Sheldon G. Adelson Medical Research Foundation, la Fondazione di JPB, la Novartis Istituto di ricerca di base, generosi contributi da Vincent e Stella Coates e il Damon Runyon Cancer Research Foundation (DRG-2125-12).

Tutte le procedure che coinvolgono roditori conformano alle linee guida della Stanford University, conformi alle politiche e nazionali e le leggi dello stato. Tutte le procedure di animali sono state approvate dal pannello di amministrazione dell'Università di Stanford sulla cura degli animali di laboratorio.
Nota: Tutte le composizioni di soluzione e buffer sono fornite nella Tabella materiali.
1. preparare una piastra di Petri per CD11b Immunopanni...

     

<ol>
	<li>Colonna, M., Butovsky, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+Function+in+the+Central+Nervous+System+During+Health+and+Neurodegeneration.">Microglia Function in the Central Nervous System During Health and Neurodegeneration.</a> Annu Rev Immunol. 35, 441-468 (2017).</li><li>Wu, Y., Dissing-Olesen, L., MacVicar, B. A., Stevens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia:+Dynamic+Mediators+of+Synapse+Development+and+Plasticity.">Microglia: Dynamic Mediators of Synapse Development and Plasticity.</a> Trends Immunol. 36 (10), 605-613 (2015).</li><li>Lou, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+receptor+P2RY12-dependent+microglial+closure+of+the+injured+blood-brain+barrier.">Purinergic receptor P2RY12-dependent microglial closure of the injured blood-brain barrier.</a> Proc Natl Acad Sci U S A. 113 (4), 1074-1079 (2016).</li><li>Haynes, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+P2Y12+receptor+regulates+microglial+activation+by+extracellular+nucleotides.">The P2Y12 receptor regulates microglial activation by extracellular nucleotides.</a> Nat Neurosci. 9 (12), 1512-1519 (2006).</li><li>Gosselin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+environment-dependent+transcriptional+network+specifies+human+microglia+identity.">An environment-dependent transcriptional network specifies human microglia identity.</a> Science. 356 (6344), (2017).</li><li>Lavin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophage+enhancer+landscapes+are+shaped+by+the+local+microenvironment.">Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment.</a> Cell. 159 (6), 1312-1326 (2014).</li><li>Hoeffel, G., Ginhoux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+Tissue-Resident+Macrophages.">Ontogeny of Tissue-Resident Macrophages.</a> Front Immunol. 6, 486 (2015).</li><li>Bennett, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+tools+for+studying+microglia+in+the+mouse+and+human+CNS.">New tools for studying microglia in the mouse and human CNS.</a> Proc Natl Acad Sci U S A. 113 (12), E1738-E1746 (2016).</li><li>Bohlen, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+Requirements+for+Microglial+Survival,+Specification,+and+Function+Revealed+by+Defined-Medium+Cultures.">Diverse Requirements for Microglial Survival, Specification, and Function Revealed by Defined-Medium Cultures.</a> Neuron. 94 (4), 759-773 (2017).</li><li>Herz, J., Filiano, A. J., Smith, A., Yogev, N., Kipnis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+Cells+in+the+Central+Nervous+System.">Myeloid Cells in the Central Nervous System.</a> Immunity. 46 (6), 943-956 (2017).</li></ol>

Poiché microglia fungono da cellule immunitarie sentinella del SNC, sono altamente sensible a reagire ai cambiamenti ambientali; di conseguenza, molta cura è necessaria per ridurre le risposte infiammatorie all'interno delle cellule durante il loro isolamento e cultura8. Questa operazione viene eseguita in questo protocollo attraverso velocità e temperatura. Ogni qualvolta possibile riduce notevolmente l'attivazione, quindi tutte le fasi di centrifugazione prendere posto a 4 ° C, mantenendo le...

Come i macrofagi del parenchima CNS, microglia interagire con una vasta gamma di circuiti neuronali e gliali reti di segnalazione. Essi svolgono un ruolo fondamentale nello sviluppo e nella omeostasi del cervello attraverso potatura sinaptica, liquidazione delle cellule apoptotiche e transitorie interazioni con processi neuronali1,2. Le microglia sono primi radar-risponditore a lesioni neurologiche, estendendo le proprie procedure lunghe e sottili ai siti di lesione per coordinare le risposte infiammatorie e limitare il sanguinamento3,4. Cambiamenti nella morfologia microglial e funzione sono onnipresenti nelle lesioni di CNS sia acute che croniche, e microglia espositivo alterata morfologia, localizzazione ed espressione dei mediatori infiammatori in una vasta gamma di Stati di malattia1. Gli studi genetici umani indicano che le mutazioni che alterano il rischio per le malattie neurodegenerative sono espressi spesso prevalentemente o esclusivamente da microglia nel SNC intatto, che punta a un ruolo critico di microglia nella patogenesi della malattia o la progressione5 . Dato loro risalto nella ferita e nella malattia, favorire la comprensione della biologia microglial è una priorità per lo sviluppo di nuovi approcci terapeutici.
Molti avanzamenti critici per la comprensione della biologia microglial sono sorti estrapolando meccanismi scoperti negli studi di altre popolazioni del macrofago, inclusi i metodi di coltura, profili di espressione genica e definizioni di funzionali e tecniche / morfologica degli Stati. Anche se generalizzato del macrofago funzioni spesso giocano fuori in modo sorprendente all'interno del paesaggio di CNS, microglia sono essi stessi altamente specializzata, che esibiscono una morfologia ramificata e una firma di espressione genetica unica che li distingue da altri tessuti macrofagi6. Microglia hanno un lignaggio che è distinto dalla maggior parte degli altri macrofagi dei tessuti; Essi colonizzano SNC durante un'ondata di embrionale precoce dell'emopoiesi primitivo e auto-rinnovarsi per tutta la vita, indipendente di contributi da ematopoiesi definitivo7. La firma di espressione genica pienamente matura di microglia adulta non viene raggiunta fino alla seconda settimana postnatale8. Stimoli ambientali dal tessuto circostante ha un ruolo importante nel dettare tessuto-specifica del macrofago caratteristiche6, che nel SNC comprende limitata esposizione ai fattori ematica concesso dalla barriera emato - encefalica9.
Un ostacolo alla piena comprensione microglial contributi al CNS omeostasi e malattia è la difficoltà di ricapitolare le proprietà specializzate di microglia maturo visto nel vivo con le cellule purificate in vitro. Sono stati sviluppati molti metodi per isolare e cultura intatta microglia, ma la maggior parte degli approcci si basano su siero per sostenere la sopravvivenza delle cellule. Abbiamo dimostrato che l'aggiunta di siero, che è un reagente intrinsecamente variabile contenente una vasta gamma di molecole bioattive, è particolarmente problematico quando lavoro con microglia perché promuove una morfologia ameboide, aumento della proliferazione, e fagocitosi aumentata9 spesso visto in vivo quando microglia sono esposti a fattori di ematica dopo l'interruzione della barriera sangue - cervello. Di queste metriche, le cellule esposte al siero assomigliano microglia in lesioni o Stati di malattia, ma tali alterazioni sono ridotti quando microglia sono coltivate in mezzo di sviluppo definito contenente CSF-1 (o IL-34), TGF-β, colesterolo e selenite.
Questo protocollo fornisce dettagli per la coltura di microglia giovanile del ratto nelle circostanze senza siero, relazionati al lavoro recentemente pubblicato9. Questo protocollo è stato ottimizzato per i ratti da giorno postnatale 21-30 (P21 - P30), ma può essere adattato per isolare microglia da ratti e topi di qualsiasi età, anche se resa e redditività complessiva varia a seconda della specie e l'età dell'animale. Maximal cede e redditività ottimale è raggiunta quando si utilizza un po' immatura microglia (~ P9), con rendimenti e vitalità si assottiglia gradualmente per un po ' i livelli più bassi negli animali adulti. Microglia può anche essere isolata dai topi, ma abbiamo trovato che le cellule del ratto mostrano significativamente più alti rendimenti, attuabilità e la complessità delle morfologie ramificate, rispetto alle cellule di topo in culture senza siero. Gli animali di età compresa tra maggiore di P50 non sono state valutate con questo protocollo. Questa procedura di isolamento di immunopanning è stata ottimizzata per minimizzare i cambiamenti nei profili trascrizionali microglial durante l'isolamento e per massimizzare a valle vitalità delle cellule. Utilizzando queste tecniche e formulazioni di media, alta redditività colture primarie possono essere sostenuti per settimane. Microglia in coltura in queste condizioni presentano una morfologia altamente ramificata che coinvolgono rapida estensione e retrazione dei processi e relativamente basso tasso di proliferazione. Evidenziamo il significato di siero-esposizione su queste proprietà e discutere i punti di forza e di debolezza di questo metodo rispetto altri approcci.

<table>
	<tbody>
		<tr>
			<td>Rat: Sprague-Dawley</td>
			<td>Charles River</td>
			<td>Cat# 400</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-rat CD11b monoclonal (clone OX42)</td>
			<td>Bio-Rad</td>
			<td>Cat# MCA275R</td>
			<td>Panning: 1:1,000; Staining: 1:500</td>
		</tr>
		<tr>
			<td>Goat polycolonal anti-Iba1&#160;</td>
			<td>Abcam</td>
			<td>Cat# AB5076</td>
			<td>Staining: 1:500</td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti-Ki67&#160;</td>
			<td>Abcam&#160;</td>
			<td>Cat# AB15580</td>
			<td>Staining: 1:500</td>
		</tr>
		<tr>
			<td>Alexa Fluor Donkey anti-mouse 594</td>
			<td>Invitrogen</td>
			<td>Cat# 11055</td>
			<td>Staining: 1:500</td>
		</tr>
		<tr>
			<td>Alexa Fluor Donkey anti-goat 488</td>
			<td>Invitrogen</td>
			<td>Cat# A-21203</td>
			<td>Staining: 1:500</td>
		</tr>
		<tr>
			<td>Alexa Fluor Donkey anti-Rabbit 647</td>
			<td>Invitrogen</td>
			<td>Cat# A-31573</td>
			<td>Staining: 1:500</td>
		</tr>
		<tr>
			<td>Triton-X (detergent in ICC staining)</td>
			<td>Thermo Fisher</td>
			<td>Cat# 28313</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparan sulfate</td>
			<td>Galen Laboratory Supplies</td>
			<td>Cat# GAG-HS01</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma&#160;</td>
			<td>Cat# M3149</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone from milk solids</td>
			<td>Sigma</td>
			<td>Cat# P6838</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-&#946;2</td>
			<td>Peprotech</td>
			<td>Cat# 100-35B</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine IL-34</td>
			<td>R&#38;D Systems</td>
			<td>Cat# 5195-ML/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovine wool cholesterol</td>
			<td>Avanti Polar Lipids</td>
			<td>Cat# 700000P</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Corning&#160;</td>
			<td>Cat# 354233</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic acid</td>
			<td>Cayman Chemicals&#160;</td>
			<td>Cat# 90260</td>
			<td></td>
		</tr>
		<tr>
			<td>11(Z)Eicosadienoic (Gondoic) Acid</td>
			<td>Cayman Chemicals&#160;</td>
			<td>Cat# 20606</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein AM dye</td>
			<td>Invitrogen</td>
			<td>Cat# C3100MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium homodimer-1</td>
			<td>Invitrogen</td>
			<td>Cat# E1169</td>
			<td></td>
		</tr>
		<tr>
			<td>DNaseI</td>
			<td>Worthington</td>
			<td>Cat# DPRFS</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll PLUS</td>
			<td>GE Healthcare</td>
			<td>Cat# 17-5445-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Sigma</td>
			<td>Cat# T9935</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>Cat# 21041-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin</td>
			<td>Gibco</td>
			<td>Cat# 15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Gibco</td>
			<td>Cat# 25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl cysteine&#160;</td>
			<td>Sigma</td>
			<td>Cat# A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>Cat# 16634</td>
			<td></td>
		</tr>
		<tr>
			<td>Apo-transferrin&#160;</td>
			<td>Sigma</td>
			<td>Cat# T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>Cat# S-5261</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (high glucose)</td>
			<td>Gibco</td>
			<td>Cat# 11960-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Dapi Fluoromount-G</td>
			<td>Southern Biotech</td>
			<td>Cat# 0100-20&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine&#160;</td>
			<td>Sigma</td>
			<td>Cat# A-003-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Primaria Plates&#160;</td>
			<td>VWR</td>
			<td>Cat# 62406-456</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Stock reagents&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Apo-transferrin</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 10 mg/mL in PBS 
			Concentration used: 1:100 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>N-acetyl cysteine</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 5 mg/mL in H2O 
			Concentration used: 1:1,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 2.5 mg/mL in H2O 
			Concentration used: 1:25,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 200 &#956;g/mL in PBS 
			Concentration used: 1:100 
			Storage: -80&#176;C</td>
		</tr>
		<tr>
			<td>TGF-b2</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 2 mg/mL in PBS 
			Concentration used: 1:1,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>IL-34</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 200 &#956;g/mL in PBS 
			Concentration used: 1:1,000 
			Storage: -80&#176;C</td>
		</tr>
		<tr>
			<td>Ovine wool cholesterol</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 1.5 mg/mL in 100% ethanol 
			Concentration used: 1:1,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Heparan sulfate</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 1 mg/mL in H2O 
			Concentration used: 1:1,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Oleic acid/Gondoic acid</td>
			<td></td>
			<td></td>
			<td>Reconstitution: Gondoic: 0.001 mg/mL; Oleic: 0.1 mg/mL in 100% ethanol 
			Concentration used: 1:1,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 50 mg/mL in PBS 
			Concentration used: 1:100 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion Buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: 50 &#956;g/mL heparin in DPBS++ (PBS with Ca++ and Mg+ +) 
			Comments: Use when ice-cold</td>
		</tr>
		<tr>
			<td>Douncing Buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: 200 &#956;L of 0.4% DNaseI in 50 mL of DPBS++ 
			Comments: Use when ice-cold</td>
		</tr>
		<tr>
			<td>Panning Buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: 2 mg/mL of peptone from milk solids in DPBS++</td>
		</tr>
		<tr>
			<td>Microglia Growth Medium (MGM)</td>
			<td></td>
			<td></td>
			<td>Recipe: DMEM/F12 containing 100 units/mL penicillin, 100 &#956;g/mL streptomycin, 2 mM glutamine, 5 &#956;g/mL N-acetyl cysteine, 5 &#956;g/mL insulin, 100 &#956;g/mL apo-transferrin, and 100 ng/mL sodium selenite 
			Comments: Use ice-cold MGM to pan microglia off of immnopanning dish.</td>
		</tr>
		<tr>
			<td>Collagen IV Coating</td>
			<td></td>
			<td></td>
			<td>Recipe: MGM containing 2 &#956;g/mL collagen IV</td>
		</tr>
		<tr>
			<td>Myelin Seperation Buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: 9 mL Percoll PLUS, 1 mL 10x PBS without Ca++ and Mg++, 9 &#956;L 1 M CaCl2, 5 &#956;L 1 M MgCl2 
			Comments: Mix well after the addition of&#160; CaCl2 and MgCl2</td>
		</tr>
		<tr>
			<td>TGF-b2/IL-34/Cholesterol containing growth media (TIC)&#160;</td>
			<td></td>
			<td></td>
			<td>Recipe: MGM containing human 2 ng/mL TGF-b2, 100 ng/mL murine IL-34, 1.5 &#956;g/mL ovine wool cholesterol, 10 &#956;g/mL heparan sulfate, 0.1 &#956;g/ml oleic acid, and 0.001 &#956;g/ml gondoic acid 
			Comments: Make sure to add cholesterol to media warmed to 37 &#176;C and do not add more than 1.5 &#956;g/mL or it will precipitate out. Do not filter cholesterol-containing media. Equilibrate TIC media with 10% CO2 for 30 min- 1 hr before adding to cells to insure optimal pH.</td>
		</tr>
	</tbody>
</table>

isolation and culture of rodent microglia to promote a dynamic ramified morphology in serum-free medium

Microglia rappresentano il 5-10% di tutte le cellule del sistema nervoso centrale (SNC) e sono di disegno sempre più attenzione grazie al loro contributo durante lo sviluppo, l'omeostasi e la malattia. I macrofagi sono stati studiati in dettaglio per decenni, caratteristiche specializzate di microglia, i macrofagi residenti in tessuto del SNC, sono rimaste in gran parte misterioso, in parte a causa di limitazioni nella capacità di ricapitolare microglial maturo Proprietà nella cultura. Qui, vi illustriamo una procedura semplice per l'isolamento rapido di microglia pura da roditore cervello maturo. Descriviamo anche condizioni di coltura privo di siero che supportano alti livelli di redditività microglial nel corso del tempo. Microglia coltivate in queste condizioni definite e medie mostre elaborati processi ramificati e comportamento dinamico sorveglianza. Illustriamo alcuni effetti di esposizione sierica sulla microglia coltivate ed discutere come queste culture senza siero confrontare culture siero-esposti così come il microglia in vivo.

Questo protocollo descrive un metodo di cultura elevata purezza ramificata microglia da ratti giovani. Risultati simili possono essere ottenuti utilizzando animali immaturi, perinatali e adulti, come discusso di seguito. Poiché gli isolamenti delle cellule includono spesso sottili sfumature e molte opportunità per cellule di morire, abbiamo usato i punti di controllo di controllo di qualità per aiutare a determinare il successo alle varie fasi. Abbiamo controllato in genere sospensioni...

Watch this Scientific Journal Video about Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium at JoVE.com

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Sforzi per comprendere la funzione di microglial in dettaglio sono stati ostacolati dalla mancanza di modelli di cultura microglial che ricapitolano le proprietà di microglia maturo in vivo . Questo protocollo descrive un metodo di isolamento e coltura progettato per mantenere la sopravvivenza robusto di ratto maturo altamente ramificato microglia in condizioni definite e medie.

Isolamento e coltura di Microglia roditore per promuovere una dinamica ramificate morfologia in Medium senza siero

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

The overall goal of this cell culture protocol is to generate highly pure cultures of microglia from developing or adult rodents that can be maintained without exposure to serum. This procedure can help answer key questions in neuroscience related to microglia morphology, fibrous ketosis, and microglia's interaction with other CNS cell types. The main advantage to this protocol is that microglia can be rapidly isolated from rodent tissue of any age and cultured without exposure to serum.To remove the brain, cut the occipital condyle from the spinal cord of an animal on both sides with dissection scissors, being careful not to damage the brain. Then, cut skin along the top of the skull, and cut up one side along the parietal and frontal bone towards the nasal bone. Carefully pull back the top of the skull with forceps, quickly remove the brain, and place it into prechilled DPBS on ice.Repeat the procedure for all remaining animals. After the brains have been collected in cold DPBS, chop each brain into one cube millimeter chunks in a petri dish on ice with a cold scalpel blade and transfer to an ice cold dounce homogenizer with five to seven milliliters of ice cold douncing buffer. Next, use a loose fitting dounce homogenizer to dissociate the tissue with 10 to 20 gentle and incomplete strokes.Take care not to directly crush the tissue at the bottom of the homogenizer. Instead, impel the tissue through the space between the sides of the piston and the homogenizer. It is important to slowly homogenize the tissue with multiple rounds of douncing to preserve microglia viability.Afterward, carefully remove the piston to prevent introducing air bubbles. Allow poorly dissociated tissue chunks to settle to the bottom of the homogenizer and transfer the supernatant to a new, chilled, 50 milliliter conical tube. Subsequently, replace the removed volume with fresh douncing buffer and repeat the dissociation procedure for a total of three to four rounds, or until all tissue has been dissociated.In this procedure, add ice cold douncing buffer to the 50 milliliter conical tube containing the cell suspension and adjust the total volume to 33.5 milliliters. Next, add 10 milliliters of MSB to the cell suspension and mix thoroughly by inverting the tube several times. This will result in a 23%final concentration of MSB in a volume of 43.5 milliliters.After that, centrifuge the cells for 15 minutes at 500 x g at 4 degrees Celsius with slow braking. Then, remove the top layer of myelin and debris and the supernatant with a pipette. Take care when removing the top layer to ensure as much of it is removed as possible.Following that, resuspend the cell pellet in 12 milliliters of panning buffer. Gently triturate the cell suspension to break up any remaining clumps of cells. In this step, pass the cell suspension through a 70 micrometer cell strainer to remove large debris or cell clumps.Rinse the OX42 coated panning dish three times with DPBS. Don't allow the plate to completely dry between washes. Pour off the last DPBS wash and apply the filtered cell suspension to the panning dish.Gently swirl the plate to distribute the cells. Then, incubate the plate on a flat surface at room temperature for 20 minutes to allow cells to adhere. Do not incubate longer than 20 minutes or cells will become very difficult to recover from the dish.Afterward, rinse the panning dish with DPBS 10 times to remove nonadherent cells. Microglia will be firmly attached to the plate, so swirl the plate with each rinse to ensure the removal of other nonadherent cells. Pour off the last DPBS wash and replace with 15 milliliters of DPBS and 200 microliters of trypsin.To trypsinize the cells, incubate the dish for less than or equal to 10 minutes at 37 degrees Celsius. After 10 minutes of trypsinization, microglia will still be stuck to the plate. Pour off the mixture and gently wash the plate two times with DPBS to remove the trypsin.Then, replace with 12 milliliters of ice cold MGM. Subsequently, place the panning dish on ice for two minutes to help weaken cell and substrate interaction and make sure that the dish is flat to prevent areas of the panning dish from drying out. After that, pipette vigorously with a 10 milliliter pipette and pipette controller on high speed to recover cells from the panning dish.Draw a 16 by 16 grid with the stream of liquid from the pipette to try and remove all of the cells. It is important to find a balance between overall cell recovery and the health of your culture. Repeated pipetting of the cell supernatant against the panning dish will result in reduced viability of your culture.Check the cells under a microscope at 20x magnification to make sure that they have detached from the plate. Mark the spots on top of the dish where cells are stuck and repeat pipetting in those areas. Collect the cell suspension and allocate three to four milliliters of supernatant in each 15 milliliter conical tube.Spin it for 15 minutes at 500 x g at four degrees Celsius with slow braking. After 15 minutes, aspirate the supernatant, leaving 0.5 milliliters of MGM with the cell pellet. Then, resuspend each pellet in remaining MGM and pull the cells from all the tubes.Following that, count the cells with a hemocytometer. Now, plate 15 microliters of collagen IV coating directly in the center of a 24 well anionic cationic coated tissue culture plate and incubate for 15 minutes at 37 degrees Celsius. After counting the cells with a hemocytometer, dilute them to 2.3 x 10 to the five per milliliter in MGM.Aspirate collagen IV spot and immediately plate 15 microliters of cell suspension to this spot to yield 3.5 x 10 to the three cells per spot. Next, incubate for five to ten minutes at 37 degrees Celsius to allow the cells to adhere. Then, gently add 500 microliters of CO2 equilibrated TIC to the well.If plating on cover slips, first coat the sterile glass cover slips with 10 micrograms per milliliter PDL in water for one hour at room temperature. Then, wash them three times with water and let them dry in a hood under UV light before proceeding with spot plating on the cover slips. In order to demonstrate successful isolation and culture of highly pure microglia, we isolated microglia from P21 rats and cultured these cells in TIC or TIC supplemented with 10%FCS for eight days.Cells were then fixed and stained with microglial markers and proliferation markers. Cells cultured in TIC show a ramified morphology, low levels of proliferation, and expression of microglia markers. In comparison, cells cultured in TIC supplemented with 10%FCS also stained for microglial markers, but show high levels of proliferation and amoeboid morphology, properties often seen in activated microglia.In addition to staining for cell markers, RNA-Seq on freshly isolated cells can provide a powerful and sensitive tool for evaluating the RNA profile of the cells and can help inform the starting purity of cultures. CD11b micropanning typically results in a small percentage of CD11b positive neutrophils and monocyte carryover, in addition to CD11b positive microglia. These cells will die in TIC after a few days, but can complicate analysis of earlier timepoints.Here is a time lapse movie of P21 rat microglia after 14 DIV in TIC. After two weeks, serum free cultures show dynamic surveillance behavior with rapid process extensions and retractions throughout the dish. Here is a time lapse movie of P21 rat microglia after five DIV in TIC before and after exposure to fetal calf serum.A rapid morphological change is evident after serum exposure. Once mastered, this protocol can be done in four to five hours, if performed properly. Following this procedure, other assays to monitor phagocytosis, chemotaxis, survival, cytokine release, transcriptional responses to stimuli, can be performed in order to answer additional questions about microglia function.After watching this video, you should have a good understanding on how to rapidly isolate microglia from adult rodent tissue and culture them under serum free conditions.

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