Finanziato dal programma intramurale presso l&#39;Università Uniformed Services.

1. La dissezione del tessuto neonatale cervello di ratto <ol><li> Freddo Leibovitz L-15 mezzi condizionati (Leibowitz L-15 + 0,1% BSA + 1% Pen / Strep) a 4 ° C. Terreni di coltura caldi (DMEM + 10% FBS + 1% di penicillina / streptomicina) a 37 ° C. Preparare 4 60x15 mm piastre Petri con 4-5 ml del condizionato L-15 supporti su ghiaccio. Sterilizzare tutti gli strumenti chirurgici con il 100% di etanolo. </li><li> Sciacquare i cuccioli P2 rap neonatali con il 70% di etanolo. (Cuccioli di ratto neonato di età compresa tra P1-P5 può essere utilizzato in questo protocollo.) </li><li> Rapidamente decapitare un topo cucciolo con sterili forbici affilate e rilasciare la testa immediatamente in etanolo al 70%. Ripetere l&#39;operazione per un totale di 5 cuccioli di ratto poi trasferire teste in soluzione salina. </li><li> Rimuovere i cervelli interi dalle teste e posto in una capsula di Petri con 4 - 5 ml di soluzione L-15 (Leibowitz L-15 + 0,1% BSA + 1% Pen / Strep) su ghiaccio. </li><li> Ripetere le procedure di 1,2 - 1,4 per le restanti cuccioli nella cucciolata. </li><li> Rimuovere le meningi dal cervello e trasferire il destessuto cerebrale IRED (cioè, corteccia, cervelletto, cervello intero, ecc) in una capsula Petri nuovo con 4-5 ml di soluzione di L-15 (Leibowitz L-15 + 0,1% BSA 1% + Pen / Strep) su ghiaccio. </li></ol> 2. Preparazione mista popolazione di cellule gliali <ol><li> Senza danneggiare il tessuto cerebrale con le forbici o una lama, trasferire il tessuto con una pipetta 10 ml ad una sterile tubo da 50 ml. </li><li> Centrifugare conico a 2.500 RCF per 5 min a 4 ° C. </li><li> Aspirare il surnatante. Aggiungere 4-5 ml di fresche L-15 media (Leibowitz L-15 + 0,1% BSA + 1% Pen / Strep). </li><li> Pipettare il tessuto su e giù per 10 volte con una pipetta sterile 10 ml. </li><li> Mettere un filtro cella (100 pori micron) su un nuovo tubo da 50 ml. Pipettare tessuto su e giù una volta con un ml pipetta sterile 5 e con la pipetta a filo del filtro cella, erogare il materiale attraverso il filtro cella nel tubo conico. </li><li> Sciacquare il filtro cella con 4-5 ml di fresche L-15 media (Leibowitz L-15 + 0,1% BSA Pen + 1% / Strep). </li><li> Centrifuga conica con cellule tese a 2.500 RCF per 5 min a 4 ° C. </li></ol> 3. Placcatura e manutenzione di culture miste cellule gliali <ol><li> Per ciascun cervello di ratto cucciolo trasformati, preparare 1 T-75 sterili pallone aggiungendo 12 ml di coltura supporti (DMEM + 10% FBS + 1% di penicillina / streptomicina) in ciascuna beuta. </li><li> Aspirare il supernatante dalle cellule pellettate e aggiungere 5-6 ml di terreni di coltura per il pellet cellulare. Pipettare su e giù per 10 volte con una pipetta 10 ml. </li><li> Trasferire un ugual volume di sospensione cellulare per ogni T-75 pallone. </li><li> Incubare matracci in 5% CO 2 incubatore a 37 ° C per un totale di 1-3 settimane. </li><li> Cilindri devono essere prima incubate per 5 giorni, e il quinto giorno, sostituire i terreni di coltura in ciascun pallone con 12 ml di mezzo fresco e tornare alla incubatrice. Poi, ogni 3 giorni, sostituire i mezzi condizionati in ogni pallone con 12 ml di mezzi freschi per ottenere confluenza. Questo deve essere farene molto attentamente senza toccare il fondo dei cilindri in cui le cellule si attaccano. </li></ol> 4. Isolamento e placcatura di primaria Microglia <ol><li> Dopo misti culture gliali sono completamente confluenti, rimuovere fiaschi dall&#39;incubatore e coprire i tappi pallone con parafilm per evitare lo scambio di gas con l&#39;aria dell&#39;ambiente. </li><li> Agitare palloni a 100 rps (Companion Lab SI-600, Jeio Tech) per 1 ora a 37 ° C. </li><li> Raccogliere i media da tutti i palloni con una pipetta 10 ml senza interrompere lo strato di astrociti sulla superficie pallone. Posizionare il supporto in provette da 50 ml. Aggiungi mezzi freschi ai fiaschi e fiaschi di ritorno alla incubatrice. </li><li> Centrifugare tubi conici a 2.500 RCF per 5 minuti a 4 ° C. </li><li> Aspirare il surnatante da tutte le provette coniche. Pellets cellulari sono alte cellule microglia purezza. Pellet risospendere in 1 ml di placcatura mezzi microglia (FES DMEM + 10% + 1% di penicillina / streptomicina). </li><li> Conta la densità delle cellule nei media risospeso utilizzandoun emocitometro. </li><li> Aggiungere un volume appropriato di terreni in piastra microglia per ottenere un 2 x 10E5 cellule per densità di ml. Piastra come appropriato per l&#39;analisi sperimentale. </li><li> Lasciare microglia di allegare tutta la notte. </li></ol> 5. Risultati rappresentativi Il protocollo descritto sopra i risultati in alte culture microglia purezza primarie. Come stabilito dal immunoistochimica per un macrofago / microglia marcatore della cella specifico (Iba1), placcati culture microglia sono&gt; 90% (Figura 2). Inoltre, per la colorazione marcatori cellulari astrociti, oligodendrociti e neuroni specifici dimostra la contaminazione minima (Figura 2). Dopo aver stabilito miste colture di cellule gliali, microglia può essere isolato agitando fino a 4 volte successive. L&#39;isolamento iniziale microglia produrrà circa 2,2 x 106 cellule / ml o circa 9 milioni di cellule per 10 palloni e il rendimento atteso diminuisce con ogni sha successiveke. Microglia isolato da questo metodo sono stati utilizzati con successo per indagare la capacità fagocitosi, funzione come la produzione di ossido nitrico, e la tossicità neuronale (Figura 3) 13-14. <img alt="Figura 1" src="/files/ftp_upload/3814/3814fig1.jpg" /> Figura 1. Panoramica del protocollo per la preparazione di colture miste di cellule gliali e l&#39;isolamento della microglia primaria. <img alt="Figura 2" src="/files/ftp_upload/3814/3814fig2.jpg" /> Figura 2. Isolamento della microglia elevata purezza, come accertato mediante analisi immunoistochimica utilizzando microglia (Iba-1, rosso), astrociti (GFAP, verde), neurone (NeuN, rosso) e degli oligodendrociti (CC1, rosso) specifici marcatori. Colorazione per il DNA delle cellule in coltura di DAPI (blu) viene anche mostrato. <img alt="Figura 3" src="/files/ftp_upload/3814/3814fig3.jpg" /> Figura 3. Microglia isolato nel metodo descriletto sono stati utilizzati in un certo numero di saggi, inclusi, ma non limitati a, saggi fagocitosi, saggi funzionali e studi di neurotossicità. In questi studi, abbiamo trovato che la microglia (Iba-1 positivo, verde), quando esposto a controllare (A) o LPS (B) supporti trattati, aumentare la fagocitosi di fluorescente perle di lattice (rosso), che possono essere quantificati ( C). Inoltre, LPS (1ng/ml) trattamento può aumentare la produzione di ossido nitrico in microglia (D). Infine, via sia Transwell insert-separati o diretti microglia-neuroni culture, abbiamo trovato che la microglia incubate con LPS possono indurre la morte delle cellule neuronali come misurato dalla lattato deidrogenasi (LDH) release (E, F).

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Neurochem. 76, 846-854 (2001).</li><li>Ni, M., Aschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+rat+primary+microglia:+isolation,+culturing,+and+selected+applications.">Neonatal rat primary microglia: isolation, culturing, and selected applications.</a> Curr. Protoc. Toxicol. Chapter 12, Unit 12.17 (2010).</li><li>Hassan, N. F., Rifat, S., Campbell, D. E., McCawley, L. J., Douglas, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+flow+cytometric+characterization+of+newborn+mouse+brain-derived+microglia+maintained+in+vitro.">Isolation and flow cytometric characterization of newborn mouse brain-derived microglia maintained in vitro.</a> J. Leukoc. Biol. 50, 86-92 (1991).</li><li>Hassan, N. F., Prakash, K., Chehimi, J., McCawley, L. J., Douglas, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+newborn+rabbit+brain-derived+microglia.">Isolation and characterization of newborn rabbit brain-derived microglia.</a> Clin. Immunol. Immunopathol. 59, 426-435 (1991).</li><li>Hassan, N. F., Campbell, D. E., Rifat, S., Douglas, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+human+fetal+brain-derived+microglia+in+in+vitro+culture.">Isolation and characterization of human fetal brain-derived microglia in in vitro culture.</a> Neuroscience. 41, 149-158 (1991).</li><li>Frank, M. G., Wieseler-Frank, J. L., Watkins, L. R., Maier, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+highly+enriched+and+quiescent+microglia+from+adult+rat+hippocampus:+immunophenotypic+and+functional+characteristics.">Rapid isolation of highly enriched and quiescent microglia from adult rat hippocampus: immunophenotypic and functional characteristics.</a> J. Neurosci. Methods. 151, 121-130 (2006).</li><li>Byrnes, K. R., Stoica, B., Loane, D. J., Riccio, A., Davis, M. I., Faden, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabotropic+glutamate+receptor+5+activation+inhibits+microglial+associated+inflammation+and+neurotoxicity.">Metabotropic glutamate receptor 5 activation inhibits microglial associated inflammation and neurotoxicity.</a> Glia. 57, 550-560 (2009).</li><li>Loane, D. J., Stoica, B. 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Mentre questo protocollo viene normalmente utilizzato per produrre microglia puro e sano per esperimenti di ricerca, un attento esame degli aspetti tecnici durante parti critiche della procedura minimizzare la variabilità nel microglia isolato. In primo luogo, durante la dissezione di tessuto cerebrale di ratti neonati, lavorando in modo tempestivo è necessario minimizzare il danno ipossico ischemico e al tessuto. Tuttavia, è anche importante per rimuovere completamente il rivestimento meningeo dal cervello durante l...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> 60 mm x 15 mm Scatole Petri </td><td> Fisher marchio </td><td> 0875713A </td><td></td></tr><tr><td> Sharp Forbici per dissezione </td><td> Strumenti di Scienze Belle </td><td> 14094-11 </td><td></td></tr><tr><td> Dumont # 7b pinze standard suggerimenti, curvi, 11cm </td><td> Strumenti di Scienze Belle </td><td> 11270-20 </td><td></td></tr><tr><td> Dumont # 5 pinze standard suggerimenti, dritte, 11 cm </td><td> Strumenti di Scienze Belle </td><td> 11251-10 </td><td></td></tr><tr><td> Provette da 50 ml coniche </td><td> VWR </td><td> 89039-656 </td><td></td></tr><tr><td> 5 ml pipette sierologiche </td><td> Grenier Bio One </td><td> 606180 </td><td></td></tr><tr><td> 10 ml pipette sierologiche </td><td> Grenier Bio One </td><td> 607180 </td><td></td></tr><tr><td> 100 um sterile nylon filtro cella </td><td> Falco </td><td> 35-2360 </td><td></td></tr><tr><td> 75 cm 2 di coltura di tessuto fiasche </td><td> Corning </td><td> 430641 </td><td></td></tr><tr><td> Mezzo essenziale minimo di Dulbecco (DMEM) </td><td> Gibco (Invitrogen) </td><td> 31053-028 </td><td></td></tr><tr><td> Leibovitz L-15 </td><td> Gibco (Invitrogen) </td><td> 11415064 </td><td></td></tr><tr><td> Siero fetale bovino </td><td> Gibco (Invitrogen) </td><td> 16000-036 </td><td></td></tr><tr><td> Fetale siero equino </td><td> Pescatore </td><td> SH3007402 </td><td></td></tr><tr><td> Penicillina-Streptomicina </td><td> Gibco (Invitrogen) </td><td> 15140163 </td><td></td></tr><tr><td> 100% etanolo </td><td> La Graham Warner Società </td><td> 64-17-5 </td><td></td></tr><tr><td> Soluzione tampone fosfato, 10X, pH 7.4 </td><td> Qualità biologica, inc. </td><td> 119-069-131 </td><td> 1X in sterile, acqua distillata </td></tr><tr><td> Sacchetti di Biohazard </td><td> VWR </td><td> 14220-028 </td><td></td></tr><tr><td> Emocitometro </td><td> Hausser Scientific </td><td> 1492 </td><td></td></tr><tr><td> 6 e piastre di coltura cellulare con superficie Cellbind </td><td> Corning </td><td> 3335 </td><td></td></tr></tbody></table>

primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue

Conto Microglia per circa il 12% della popolazione cellulare totale nel cervello dei mammiferi. Mentre i neuroni e astrociti sono considerati i principali tipi di cellule del sistema nervoso, microglia svolgere un ruolo importante nella fisiologia normale del cervello attraverso il monitoraggio del tessuto per detriti e agenti patogeni e di mantenere l&#39;omeostasi nel parenchima via 1,2 attività fagocitaria. Microglia vengono attivati ​​nel corso di una serie di condizioni di pregiudizio e malattia, tra cui le malattie neurodegenerative, lesioni cerebrali traumatiche, e l&#39;infezione del sistema nervoso 3. In queste condizioni attivanti, microglia aumentare la loro attività fagocitaria, subire una modifica morpohological e proliferativi, e attivamente secernono le specie reattive dell&#39;ossigeno e dell&#39;azoto, pro-infiammatorie chemochine e citochine, spesso l&#39;attivazione di un loop paracrino o autocrino 4-6. Come queste risposte microgliali contribuire alla patogenesi della malattia in condizioni neurologiche, ricerca incentrata sulla microglia è garantito. A causa della eterogeneità cellulare del cervello, è tecnicamente difficile ottenere sufficiente materiale microglia campione con elevata purezza Durante gli esperimenti in vivo. La ricerca attuale sulle funzioni neuroprotettive e neurotossici di microglia richiedono un metodo di routine tecnica per generare costantemente microglia puro e sano con rendimento sufficiente per lo studio. Presentiamo, nel testo e video, un protocollo per isolare puro microglia primaria da colture miste glia per una varietà di applicazioni a valle. In breve, questa tecnica utilizza il tessuto cerebrale dissociata da ratti neonati per la produzione di colture miste di cellule gliali. Dopo le colture miste gliali raggiungano la confluenza, microglia primari sono meccanicamente isolato dalla coltura mediante una durata breve agitazione. Le microglia vengono quindi piastrate ad elevata purezza per studio sperimentale. Il principio e il protocollo di questa metodologia sono statidescritto in letteratura 7,8. Inoltre, le metodologie alternative per isolare microglia primaria sono ben descritte 9-12. Tessuto cerebrale omogeneizzati possono essere separate mediante centrifugazione in gradiente di densità a cedere primaria microglia 12. Tuttavia, la centrifugazione è di lunghezza moderata (45 min) e può causare danni cellulari e attivazione, così come, causano arricchito microglia e altre popolazioni cellulari. Un altro protocollo è stato utilizzato per isolare microglia primaria in una varietà di organismi da prolungato (16 ore) agitando mentre nella cultura 9-11. Dopo aver agitato, il surnatante dei media viene centrifugato per isolare microglia. Questo metodo di isolamento più due fasi possono anche turbare la funzione e l&#39;attivazione della microglia. Si utilizzano principalmente il seguente protocollo di isolamento microglia nel nostro laboratorio per una serie di ragioni: (1) primaria microglia simulare in biologia vivo più fedelmente rispetto alle linee di cellule microglia immortalati roditori, (2) Rottura nominale meccanica minimizza potenziale disfunzione cellulare o l&#39;attivazione, e (3) resa sufficiente può essere ottenuta senza passaggio delle colture miste di cellule gliali. È importante notare che questo protocollo utilizza tessuto cerebrale da ratti neonati per isolare microglia e che utilizzando ratti anziani isolare microglia possono influenzare notevolmente la resa, stato di attivazione, e le proprietà funzionali della microglia isolato. Ci sono prove che l&#39;invecchiamento è collegato con disfunzione microglia, neuroinfiammazione aumentata e patologie neurodegenerative, studi precedenti hanno quindi utilizzati ex vivo adulti microglia per comprendere meglio il ruolo della microglia nelle malattie neurodegenerative in cui l&#39;invecchiamento è importante parametro. Tuttavia, ex vivo microglia non possono essere mantenute in coltura per periodi di tempo prolungati. Pertanto, mentre questo protocollo prolunga la vita della microglia in coltura primaria, va osservato che la microglia comportarsi differently da adulto microglia e studi in vitro deve essere attentamente considerato quando tradotto in un ambiente vivo.

Watch this Scientific Journal Video about Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue at JoVE.com

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

Isolamento microglia primaria dalla eterogeneità cellulare del cervello è essenziale per studiare il loro ruolo sia in condizioni fisiologiche e patologiche. Questo protocollo descrive un isolamento meccanico e mista tecnica di coltura cellulare che fornisce alta resa ed elevata purezza, cellule vitali primarie per microgliali<em&gt; In vitro</em&gt; Studio e applicazioni a valle.

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 ...

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JoVE Journal

Immunology and Infection

39.1K Views.  Uniformed Services University. Isolamento microglia primaria dalla eterogeneità cellulare del cervello è essenziale per studiare il loro ruolo sia in condizioni fisiologiche e patologiche. Questo protocollo descrive un isolamento meccanico e mista tecnica di coltura cellulare che fornisce alta resa ed elevata purezza, cellule vitali primarie per microgliali In vitro Studio e applicazioni a valle.

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

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Un metodo semplice ed efficace per isolare i macrofagi da Mixed colture primarie di cellule del fegato degli adulti

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Ricerca

Immunologia e infezioni

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

Generazione di culture Raft organotipiche da cheratinociti umani primari

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Coltura Microglia dal Sistema Nervoso Centrale neonatale e adulti

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

Isolamento della corticale Microglia con Immunofenotipo conservato e funzionalità dal murini neonati

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Isolamento e iniezione endovenosa di Murine derivate dal midollo osseo monociti

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

Isolamento delle cellule di Sertoli e peritubulari cellule da Rat testicoli

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Isolamento e analisi citofluorimetrica delle cellule immunitarie dal cervello ischemica mouse

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

L&#39;induzione di una risposta infiammatoria in colture primarie epatociti di topi

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

Valutazione Stress cellulare e l'infiammazione nei Nuclei di cervello che secerne l'ossitocina discreti nel ratto neonatale prima e dopo la prima poppata di colostro

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa parasites.

Isolamento concomitante di astrociti primari e microglia per l'infezione da parassiti di Protozoi

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central ...