Name: isolation and culture of primary neurons and glia from adult rat urinary bladder
Uploaded: 2020-05-23T14:45:00.000+00:00
Duration: 6 min 18 s
Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder at JoVE.com

Questo studio è stato supportato dalla National Natural Science Foundation of China (Grant n. 81673676) e dal Dongguan Science and Technology Bureau (Grant n. 2019622101002). Gli autori ringraziano la Dott.ssa Maryrose Sullivan (Assistente professore in Chirurgia, Harvard Medical School) per la consulenza tecnica.

Tutti i protocolli sperimentali e le procedure animali sono conformi alle linee guida del principio etico del Consiglio Nazionale delle Ricerche.
1. Preparazione dei materiali
<ol><li>Sterilizzare tutti gli strumenti e ddH2O utilizzando un'autoclave prima di eseguire l'esperimento. Gli strumenti includono ma non sono limitati a forbici chirurgiche, forbici oftalmiche, forcep, divisori a nucleo cucchiai, piatti di vetro (60-100 mm di diametro) e rompisceppi di vetro.</li>
	<li>Preparare la soluzione di Krebs come segue(tabella 1):Sciogliere tutte le sostanze chimiche insieme a ddH2O prima dell'uso. Sciogliere CaCI2 separatamente e aggiungere lentamente nella soluzione mista mescolando per evitare sedimenti.</li>
	<li>Preparare il supporto rinse, costituito da mezzi F12 con siero bovino fetale al 10% (FBS) e 1% antibiotico /antimicotico (100x). Aggiungere 5 mL di FBS e 0,5 mL di antibiotico/antimicotico a 44,5 mL di mezzi F12.</li>
	<li>Preparare il supporto neuronale A, che comprende mezzi neurobasali A con 2% B-27, 1% FBS, 1% L-glutammina, 1% antibiotico/antimicotico (100x) e 0,1% fattore neurotrofico derivato da glia (GDNF). Aggiungere 200 μL di B-27, 100 μL di FBS, 100 μL di L-glutammina, 100 μL di antibiotico/antimicotico (100x) e 10 μL di GDNF (10 μg/mL) a 9,5 mL di mezzi neurobasali A. NOTA: Preparare il supporto neuronale A entro una settimana dall'uso per garantire la freschezza di B-27, L-glutammina e GDNF.</li>
	<li>Preparare il supporto neuronale B utilizzando la stessa procedura della preparazione dei mezzi neuronali A ma senza aggiungere FBS.</li>
	<li>Preparare la soluzione di digestione 1 sciogliendo 20 mg di collagenasi di tipo II, 6 mg di albumina di siero bovino e 200 μL di antibiotico/antimicotico (100x) in 10 mL di soluzione Krebs stabile all'ossigeno.</li>
	<li>Preparare la soluzione di digestione 2 diluendo 1 mL di 0,25% di tripside con 4 mL della soluzione salina bilanciata di Hank.</li>
	<li>Preparare una piastra con copripavimenti rivestiti.<ol>
<li>Utilizzare le forcep in un banco di flusso laminare quando si posizionano le coverlips di vetro in una piastra di coltura da 48.</li>
		<li>Diluire la poli-D-llysina con ddH2O ad una concentrazione di 0,1 mg/mL come stock di poli-D-lsina. Conservare il titolo a -20 °C e scongelare prima dell'uso.</li>
		<li>Aggiungere 40 μL di poli-D-lisina sopra ogni coverlip e incubare la soluzione a temperatura ambiente per 10 minuti. NOTA: Per diverse piastre con diverse aree basali, regolare la concentrazione a 4 μg/cm2. Ad esempio, se l'area basale di un pozzo da una piastra di 24 po 'è 2 cm2, allora dovremmo trasferire 80 μL di poli-D-lisina in un pozzo. Ogni cm2 conterrebbe 4 μg di poli-D-lsina.</li>
		<li>Rimuovere la poli-D-lisina nella piastra a 48 po' e sciacquare le coperture con ddH2O tre volte.</li>
		<li>Asciugare la piastra in una cappa di flusso laminare per almeno 30 minuti per assicurarsi che la piastra sia anidra.</li>
		<li>Conservare la piastra a 4 °C prima del rivestimento di laminina per 1 giorno al massimo. Conservare la piastra a -20 °C è preferibile per lo stoccaggio a lungo termine.</li>
		<li>Scongelare laminina a 4 °C. Diluire laminina con ddH2O ad una concentrazione di 50 μg/mL come stock di laminina. Conservare il titolo a -20 °C e scongelare a 4 °C prima dell'uso.</li>
		<li>Utilizzare una pipetta per trasferire 100 μL di laminina diluita nella parte superiore di ogni coverlip e incubare la laminina a 4 °C per 1 h. NOTA: Per diverse piastre con diverse aree basali, regolare la concentrazione a 5 μg/cm2. Ad esempio, se l'area basale di un pozzo da una piastra di 24 po 'è 2 cm2, trasferire 200 μL di laminina diluita in un pozzo. Ogni cm2 conterrebbe 5 μg di laminina.</li>
		<li>Rimuovere la soluzione di laminina e sciacquare i copriscippi con ddH2O una volta. Eseguire questa operazione sul bordo del coperchio per evitare il raschiamento. NOTA: I copripasci rivestiti potrebbero essere conservati a 4 °C per al massimo 2 settimane.</li>
	</ol>
</li>
</ol>2. Raccolta vescica
<ol><li>Ottieni ratti Sprague-Dawley di cinque settimane.</li>
	<li>Infondere carbogeno (95% ossigeno, 5% CO2)nella soluzione Krebs per almeno 30 minuti in un bagno di ghiaccio per raggiungere uno stato di ossigeno stabile e un livello di pH.</li>
	<li>Dopo l'eutanasia tramite lussazione cervicale, immergere i ratti in 75% di etanolo per 30 s per la sterilizzazione.</li>
	<li>Posizionare i ratti su un asciugamano chirurgico sterilizzato ed esporre il loro addome. Apri la cavità addominale e rivela la vescica con una serie di forbici e forcelle.</li>
	<li>Sollevare delicatamente la vescica e tagliare la vescica dal collo della vescica con un altro set di forbici e forcelle per evitare la contaminazione incrociata. Posizionare rapidamente la vescica nella soluzione Krebs stabile all'ossigeno freddo per migliorare la sopravvivenza cellulare. NOTA: Una volta rimossa la vescica, eseguire rapidamente le seguenti operazioni per migliorare la prospettiva di sopravvivenza dei neuroni.</li>
	<li>Aggiungere la soluzione Krebs in tre piatti di vetro e rompisbi.</li>
	<li>Preparare piatti in vetro e rompisbi con soluzione Krebs in un bagno di ghiaccio per il preraffreddamento.</li>
	<li>Contrassegnare questi contenitori con i numeri da 1 a 3 in modo corrispondente per evitare confusione.</li>
	<li>Abbina ogni piatto di vetro a forcep e un divisore a nucleo cucchiaio.</li>
	<li>Nel piatto di vetro 1, aprire la vescica con forbici oftalmiche e dispiegarla con forcelle e un divisore a nucleo cucchiaio.</li>
	<li>Risciacquare la vescica in un rompicapo 1 e posizionare in un piatto di vetro 2.</li>
	<li>Elimina il grasso aderente sulla superficie del tessuto usando le flessce e le forbici oftalmiche nel piatto di vetro 2.</li>
	<li>Risciacquare la vescica in un rompicapo 2 e posizionarela in un piatto di vetro 3.</li>
	<li>Raschiare delicatamente la vescica usando le forcep e il divisore del nucleo cucchiaio sul piatto di vetro 3 per rimuovere gli attacchi esogeni.</li>
	<li>Risciacquare la vescica in un demolitore di vetro 3, trasferire la vescica in un tubo di centrifuga da 15 ml con 14 ml di soluzione Krebs fredda e ruotare il campione per 1 minuto a 356 x g e 4 °C.</li>
	<li>Ripetere il passaggio precedente due volte in altri due tubi con soluzione Krebs per ridurre la contaminazione.</li>
</ol>3. Digestione della vescica in due fase
<ol><li>Trasferire la vescica dal tubo di centrifuga a una fiala da 2 ml contenente 1 ml di soluzione di digestione 1. Utilizzare forbici oftalmiche per tagliare la vescica a piccoli pezzi (più piccoli di 1 mm) in soluzione.</li>
	<li>Mescolare la soluzione della vescica con 9 mL di soluzione di digestione 1 in un piatto sterile di coltura cellulare (100 mm di diametro). Eseguire la digestione del passaggio 1 in un incubatore di scuotimenti per 1 h sotto il 5% di CO2,37 °C e 200 giri/min.</li>
	<li>Dopo la digestione del passaggio 1, centrifugare la soluzione a 356 x g a 4 °C per 8 min.</li>
	<li>Posizionare la soluzione di digestione 2 in un bagno d'acqua a 37 °C per il preriscaldamento.</li>
	<li>Dopo la centrifugazione, rimuovere il supernatante che contiene la soluzione di digestione 1 e raccogliere il sedimento cellulare. È consentito un po 'di liquido rimanente. La rimozione completa della soluzione può favorire la perdita cellulare.</li>
	<li>Mescolare i sedimenti cellulari con la soluzione di digestione calda 2 in un tubo di centrifuga da 15 ml e agitare la miscela durante la digestione in un bagno d'acqua a 37 °C per 5 minuti. Non superare i 7 minuti della digestione del passaggio 2 o i neuroni periranno.</li>
	<li>Dopo la digestione, disattivare immediatamente la tripina nella miscela con 10 mL di mezzi di risciacquo a freddo. NOTA: Eseguire i seguenti passaggi a 0 °C–4 °C. Un bagno di ghiaccio potrebbe fornire tali condizioni.</li>
	<li>Raccogliere il sedimento cellulare dopo la centrifugazione a 356 x g a 4 °C per 8 min. Rimuovere il più possibile il supporto perché la tripina rimanente è dannosa per la crescita cellulare.</li>
	<li>Resopend sedimento con 3 mL di mezzi neuronali delicatamente. Assicurarsi che le bolle d'aria non vengano generate nella soluzione contenente cellule per un alto tasso di sopravvivenza.</li>
	<li>Filtrare il supporto della miscela attraverso un colino cellulare da 70 μm in un tubo di centrifuga da 50 ml.</li>
	<li>Conservare il filtrato su uno shaker a 30 giri/min in un bagno di ghiaccio per 30 minuti. Questo passaggio non è necessario ma consigliato.</li>
	<li>Raccogliere le cellule mediante centrifugazione a 356 x g a 4 °C per 8 minuti e rimorsi delicatamente il pellet cellulare in 1 mL di mezzi neuronali A.</li>
	<li>Aggiungere 500 μL di miscela cellulare in ogni pondo della piastra preparata a 48 porsi.</li>
	<li>Cellule di coltura in un incubatore a 37 °C e 5% CO2.</li>
	<li>Sostituire tutti i supporti con il supporto neuronale B in 1 h per fornire una coltura priva di siero.</li>
	<li>Cambiare metà del mezzo neuronale B ogni 3 giorni. NOTA: I neuroni sono pronti per esperimenti immunocitochimici dopo 5-7 giorni di coltura.</li>
</ol>

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Gli autori non dichiarano alcun conflitto di interessi di grande importanza.

Preparazione piastra L'uso di coverlips in vetro in piastre di coltura da 6, 12 o 48 pozzi per esperimenti di imaging immunofluorescente o di calcio è un'operazione economica e parsimonioso. Le cellule crescono bene in piastre senza coverlips durante la preparazione di colture cellulari primarie. Pertanto, le coverlips sono dispensabili in esperimenti, come la macchia occidentale o la reazione a catena della polimerasi. Inoltre, il rivestimento è un passo necessario prima di placcare le celle, con o...

Il tratto urinario inferiore ha due funzioni principali: conservazione periodica delle urine e micturizione1. Il sistema nervoso del tratto urinario inferiore (LUTNS) controlla queste funzioni ed è delicato e suscettibile a molte neuropatie, che possono essere innate (porfiria), acquisite (malattia di Lyme), secondarie agli stati di malattia (cistopatia diabetica), indotte da farmaci (cistite emorragica), chirurgia causata (resezione addominoperineale) o lesioni causate (lesione traumatica del midollo spinale)2,3,4,5,6,7. Negli studi fisiologici/patologici, gli esperimenti in vivo e in vitro sono ugualmente importanti. Mentre la ricerca in vivo sull'LUTNS è stata condotta a livello di organo, cellulare e molecolare per qualche tempo, la ricerca in vitro sui neuroni primari della vescica urinaria è quasi inesistente8,9. Sebbene il presente studio sia limitato, speriamo di essere pionieri della ricerca in questo settore in modo che altri ricercatori possano migliorarla. In questo modo, questa co-coltura può portare a una comprensione cellulare della disfunzione fisiologica nei fenotipi, come la disfunzione neuronale della vescica.
A differenza dei muscoli enterici con una chiara direzionalità delle cellule muscolari in strati discreti, i muscoli della vescica sono disorganizzati10. Pertanto, invece di staccare lo strato esterno della vescica, questo metodo propone di digerire l'intera vescica per ridurre la difficoltà di funzionamento e abbreviare i tempi di pre-elaborazione per un alto tasso di sopravvivenza cellulare.
Seguendo questo metodo, possiamo ottenere una coltura mista di neuroni e altre cellule. Le altre cellule sono indispensabili perché la loro presenza imita un ambiente in vivo11. Inoltre, tali cellule forniscono le sostanze che non sono disponibili nel mezzo.
Questo metodo prevede due passaggi per la digestione. In primo luogo, la collagenasi di tipo II viene utilizzata per idrolizzare il collagene, seguita dalla tripparina, per dissociare il tessuto nelle cellule10. In questo modo, i tessuti della vescica vengono dispersi in singole cellule e quindi crescono relativamente indipendenti. Quando la coltura dei neuroni matura, i neuroni possono essere utilizzati per l'imaging o test funzionali.

<table><tbody><tr><td>0.25% trypsin</td><td>Gibico</td><td>15050065</td><td>Enzyme digestion</td></tr><tr><td>48-well culture plate</td><td>Corning</td><td>3548</td><td>Coating dish</td></tr><tr><td>antibiotic/antimycotic</td><td>Gibico</td><td>15240062</td><td>Culture media/Rinse media</td></tr><tr><td>Anti-Glial Fibrillary Acidic Protein Antibody</td><td>Santa Cruz</td><td>sc-33673</td><td>ICC</td></tr><tr><td>B-27</td><td>Gibico</td><td>17504044</td><td>Culture media</td></tr><tr><td>BSA Fraction V</td><td>Gibico</td><td>332</td><td>Enzyme digestion</td></tr><tr><td>Choline Acetyltransferase Antibody</td><td>Abcam</td><td>ab18736</td><td>ICC</td></tr><tr><td>CO2 Incubator</td><td>Heraeus</td><td>B16UU</td><td>Cells culture</td></tr><tr><td>Collagenase type II</td><td>Sigma</td><td>2593923</td><td>Enzyme digestion</td></tr><tr><td>DMEM/F-12</td><td>Gibico</td><td>11330032</td><td>Rinse media</td></tr><tr><td>DYNLL2 Antibody</td><td>Santa Cruz</td><td>sc-13969</td><td>ICC</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibico</td><td>10100147</td><td>Culture media/Rinse media</td></tr><tr><td>Forceps</td><td>Shanghai Jin Zhong Medical Devices</td><td>1383</td><td>10 cm; Sterile operation</td></tr><tr><td>Glass breakers</td><td>Huan Qiu Medical Devices</td><td>1101</td><td>50 ml; Sterile operation</td></tr><tr><td>Glass coverslips</td><td>WHB Scientific</td><td>WHB-48-CS</td><td>Coating dish</td></tr><tr><td>Glass dishes</td><td>Huan Qiu Medical Devices</td><td>1177</td><td>100 mm; Sterile operation</td></tr><tr><td>Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 488</td><td>Southernbiotech</td><td>3050-30</td><td>ICC</td></tr><tr><td>Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 555</td><td>Southernbiotech</td><td>3050-30</td><td>ICC</td></tr><tr><td>Hoechst 33342</td><td>BD</td><td>561908</td><td>ICC</td></tr><tr><td>Laminar flow bench</td><td>Su Jie Medical Devices</td><td>CB 1400V</td><td>Sterile operation</td></tr><tr><td>Laminin</td><td>Sigma</td><td>L2020</td><td>Coating dish</td></tr><tr><td>L-glutamine</td><td>Gibico</td><td>25030081</td><td>Culture media</td></tr><tr><td>MAP-2 Antibody</td><td>Affinity</td><td>AF5156</td><td>ICC</td></tr><tr><td>Murine GDNF</td><td>Peprotech</td><td>AF45044</td><td>Culture media</td></tr><tr><td>Neurobasal-A Medium</td><td>Gibico</td><td>10888022</td><td>Culture media</td></tr><tr><td>Ophthalmic scissors</td><td>Shanghai Jin Zhong Medical Devices</td><td>J21010</td><td>12.5 cm; Sterile operation</td></tr><tr><td>Pipettes</td><td>Eppendorf</td><td>3120000240</td><td>100-1000 ul; Reagent and sample pipetting</td></tr><tr><td>Pipettes</td><td>Eppendorf</td><td>3120000267</td><td>10-100 ul; Reagent and sample pipetting</td></tr><tr><td>Poly-D-lysine</td><td>Sigma</td><td>P7280</td><td>Coating dish</td></tr><tr><td>Refrigerated centrifuge</td><td>Ping Fan Instrument</td><td>TGL-16A</td><td>Enzyme digestion</td></tr><tr><td>Shaking incubator</td><td>Haimen Kylin-Bell Lab Instruments</td><td>T8-1</td><td>Enzyme digestion</td></tr><tr><td>SLC17A9 Antibody</td><td>MBL International</td><td>BMP079</td><td>ICC</td></tr><tr><td>Spoons nucleus divider</td><td>Shanghai Jin Zhong Medical Devices</td><td>YZR030</td><td>12 cm; Sterile operation</td></tr><tr><td>Substance P Antibody</td><td>Santa Cruz</td><td>sc-58591</td><td>ICC</td></tr><tr><td>Surgical scissors</td><td>Shanghai Jin Zhong Medical Devices</td><td>J21130</td><td>16 cm; Sterile operation</td></tr><tr><td>Surgical towel</td><td>Fu Kang Medical Devices</td><td>5002</td><td>40 x 50 cm; Sterile operation</td></tr><tr><td>Synapsin-1 Antibody</td><td>CST</td><td>5297T</td><td>ICC</td></tr><tr><td>Tubulin beta Antibody(&amp;beta;-III-tubulin)</td><td>Affinity</td><td>AF7011</td><td>ICC</td></tr></tbody></table>

isolation and culture of primary neurons and glia from adult rat urinary bladder

Il tratto urinario inferiore ha due funzioni principali, vale a dire la conservazione periodica delle urine e la micturizione; queste funzioni sono mediate attraverso la neuroregolazione centrale e periferica. Sebbene sia stata condotta un'ampia ricerca sul sistema nervoso del tratto urinario inferiore, la maggior parte degli studi si è concentrata sulla cultura primaria. Questo protocollo introduce un metodo per l'isolamento e la coltura dei neuroni vescicali e degli glia dei ratti Sprague-Dawley. In questo metodo, i neuroni e glia sono stati incubati in un incubatore di 37 °C, 5% CO2 per 5-7 giorni. Di conseguenza, sono cresciuti in forme mature adatte ai successivi esperimenti di immunofluorescenza correlati. Le cellule sono state osservate morfologicamente usando un microscopio ottico. Neuroni, vescicole sinaptiche e glia sono stati identificati rispettivamente da β-III-tubulina e MAP-2, Synapsin-1 e GFAP. Nel frattempo, l'immunocitochimica è stata eseguita su diverse proteine correlate al neurotrasmettitore, come la colina acetiltransferasi, DYNLL2 e SLC17A9.

Nel processo di coltura cellulare primaria, le cellule acquisite erano rotonde con limiti luminosi e chiari prima dello stato attaccato. Man mano che i neuroni crescevano, dendriti e assoni cominciavano ad essere distinti. Dopo 5-7 giorni di coltura, i neuroni hanno raggiunto una forma matura con proiezioni lunghe, ideali per l'imaging o gli studi di funzione. Sebbene la maggior parte delle impurità e dei detriti cellulari potessero essere rimossi a causa del cambiamento dei mezzi, alcuni residui attaccati al rivestimen...

Watch this Scientific Journal Video about Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder at JoVE.com

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

Questo protocollo tenta di stabilire un protocollo ripetibile per i neuroni primari e l'isolamento degli glia dalla vescica del ratto per ulteriori esperimenti cellulari.

Isolamento e cultura dei neuroni primari e degli glia dalla vescica urinaria del ratto adulto

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

isolation-culture-primary-neurons-glia-from-adult-rat-urinary

Research

JoVE Journal

Neuroscience

3.8K Views.  Guangzhou University of Chinese Medicine. Questo protocollo tenta di stabilire un protocollo ripetibile per i neuroni primari e l'isolamento degli glia dalla vescica del ratto per ulteriori esperimenti cellulari.

Video: Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Isolamento di Neural cellule staminali / progenitrici dalla Regione periventricolare del ratto adulto e midollo spinale umano

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Ricerca

Biologia dello sviluppo

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Isolamento primario Microglia misti da colture cellulari gliali del tessuto neonatale cervello di ratto

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Immunologia e infezioni

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminar co-coltura di neuroni corticali primari di ratto e Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Neuroscienze

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Isolamento e la coltura di cellule embrionali neuronali di ratto: un protocollo veloce

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Isolamento, cultura e lungo termine Manutenzione di primaria mesencefalico neuroni dopaminergici Da Brains embrionali dei roditori

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

Preparazione delle colture gliali miste primarie da adulti mouse spinale tessuto del midollo

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Isolamento dei gangli di radice dorsale e colture primarie per lo studio del rilascio di neurotrasmettitore

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Ottenere Microglia Umana da Tessuto Cerebrale Umano Adulto

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Isolation and Culture of Primary Neurons and Glia from a Rat Urinary Bladder

Source: Wang, R. et al., <a href="https://app.jove.com/v/61177">Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder.</a> J. Vis. Exp. (2020).
This video demonstrates a technique for generating a primary culture of neurons and glia from a rat&#39;s urinary bladder. It outlines the steps involved in tissue preparation, enzymatic digestion to obtain single cells, and culturing the cells in a neuron-specific medium to establish a primary neuronal and glial culture.

Isolamento e coltura dei neuroni primari e della glia da una vescica urinaria di ratto

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Isolation of Neural Stem Progenitor Cells from the Spinal Cord Periventricular Region of an Adult Rat

Source: Mothe, A., et al., <a href="https://app.jove.com/v/52732">Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord.</a> J. Vis. Exp. (2015).
This video presents a method for isolating neural stem/progenitor cells from the periventricular tissue of the spinal cord in adult rats. The technique includes incubating the tissue fragments in a solution containing proteolytic enzymes to disrupt the extracellular matrix and release the cells. Subsequently, the released cells are plated in a medium containing specific growth factors that enhance neural stem/progenitor proliferation and promote aggregation into three-dimensional neurospheres.

Isolamento delle cellule progenitrici dello staminali neurale dalla regione periventricolare del midollo spinale di un ratto adulto

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of Dissection ...