Questo lavoro è stato finanziato da una borsa di ricerca e una borsa-in-Aid dalla Food Project Nanotecnologia del Ministero dell&#39;agricoltura, delle foreste e della pesca del Giappone.

1. Perfusione del Fegato <ol><li> Anestetizzare un adulto maschio di ratto Sprague-Dawley (a 10 a 15 settimane di età, peso corporeo 150-200 g) con un&#39;iniezione intraperitoneale di una soluzione di sodio pentobarbital (50 mg di sodio pentobarbital / kg di peso corporeo). </li><li> Posto l&#39;animale in una padella in acciaio e spray etanolo al 70% sul suo addome e torace. </li><li> Fai un piccolo taglio e di staccare la pelle. Aperto l&#39;addome dal taglio con un paio di forbici. </li><li> Confermare la posizione di vena porta, passa un filo chirurgico sotto la vena porta e liberamente si aggregano. </li><li> Clump la parte distale della vena cava inferiore e la vena mesenterica per prevenire il reflusso del sangue nel fegato. Aprire la cavità toracica tagliando il diaframma con le forbici, ed esporre il cuore. </li><li> Inserire un catetere di plastica (2 mm di diametro) nella vena porta e serrare saldamente il filo intorno al catetere. </li><li> Collegare il catetere ad un sistema di perfusione che viene caricato con soluzione di lavaggio (Ca 2 +-Mg 2 + HBSS gratuito contenente 0,5 mM EGTA, 10 HEPES mM e 4,2 mM NaHCO 3; pH 7,2) preriscaldata a 37 ° C. </li><li> Iniziare perfusione in situ a una velocità di 10 ml / min. Allo stesso tempo, fare un taglio nella parete dell&#39;atrio destro. Continua perfusione per 10 minuti. </li><li> Accendere la soluzione di perfusione alla soluzione collagenasi [HBSS contenente HEPES 10mm e 4,2 mm NaHCO 3 integrati con collagenasi di tipo I (0,05%) e inibitori della tripsina (50 mg / ml), pH 7,5], e profumato per 10 - 20 minuti a una velocità di 10 ml / min fino a quando il fegato si gonfia leggermente e mostra il segno di disintegrazione della struttura del fegato sotto membrana sierosa. </li></ol> 2. Preparazione di epatociti parenchimale ricchi frazione di cellule <ol><li> Rimuovere e trasferire con cautela il fegato in un bicchiere sterile contenente 25 ml di soluzione di collagenasi. Tritare in piccoli pezzi con le forbici. </li><li> Aggiungere 75 ml di MEM freddo nella sospensione cellulare risultante. Dopo pipettaggio dolce, filtrato attraverso un colino cellulare (100 micron) per rimuovere i tessuti connettivi e frammenti di tessuto non digerito. </li><li> Trasferire il filtrato in provette da 50 ml e centrifugare a 50 xg per 1 minuto a 4 ° C (freno off). </li><li> Eliminare attentamente il supernatante. Risospendere il pellet con MEM freddo. </li><li> Ripetere 2,4) tre volte. </li></ol> 3. Misti Cultura primaria delle cellule del fegato <ol><li> Sospendere le cellule ottenute in 2.5) nel mezzo di crescita composto da DMEM contenente 10% FCS inattivato con il calore, integrato con 100 mM β-mercaptoetanolo, 10 mg / ml di insulina, 100 mg / ml di streptomicina e 100 U / ml di penicillina, e di semi in 09:55 tessuto fiaschi cultura (superficie: 75 cm 2) ad una densità di 6,7 x 10 4 cellule / cm 2 (5 x 10 6 cellule / fiasco / 12 -15 ml di terreno). </li><li> Incubare la cultura flasksat 37 ° C in atmosfera al 5% di CO 2-95% di aria. </li><li> Sostituire il mezzo di coltura ogni 2 o 3 intervalli di giorni. </li></ol> 4. Isolamento selettivo dei macrofagi <ol><li> Dopo 12 giorni di coltura, i macrofagi proliferano sul foglio delle cellule. Queste cellule sono sospese dalle reciproche agitazione dei flaconi cultura a 80-120 colpi al minuto per 30 minuti a 37 ° C. </li><li> Trasferire il terreno di coltura in 100 mm non tessuto cultura piatti di qualità plastica. Piscina del mezzo da due T75 fiaschi in un piatto di plastica. Fiaschi cultura riempire con il mezzo di crescita e li riporta ai incubatrice. </li><li> Incubare piatti di plastica per 30 minuti a 37 ° C in atmosfera al 5% di CO 2-95% di aria. Macrofagi prontamente attribuiscono alla non-tessuti piatti di plastica di qualità della cultura in questo processo di incubazione, mentre altre cellule contaminanti non lo fanno. </li><li> Aspirare il mezzo e risciacquare delicatamente il piatto di plastica con PBS. Ripetere questa operazione tre volte. </li><li> Aggiungere 1 ml di soluzione TrypLE espresso nel piatto, e incubare per 10 minuti a 37 ° C in atmosfera al 5% di CO 2-95% di aria. </li><li> Aggiungere 9 ml del mezzo di crescita. Delicatamente togliere la macrofagi attaccato da un raschietto cellula. </li><li> Trasferire la sospensione cellulare in un tubo da 15 ml e centrifugare a 180 xg per 5 min a 25 ° C (freno). </li><li> Eliminare il supernatante e aggiungere 1 ml del mezzo di crescita. Dissociano in singole cellule pipettando vigoroso. Enumerare il numero di cellulare da un emocitometro conteggio. </li><li> Passaggi di isolamento può essere ripetuto con la stessa cultura fiaschi a intervalli di 2-3 giorni per più di due settimane. </li><li> Macrofagi isolati possono essere coltivate in terreno di coltura cui al punto 3.1. </li></ol> 5. Rappresentante Risultati Un esempio di una coltura mista primaria di cellule adulte fegato di ratto è illustrato nella figura 2. Epatociti parenchimale perdere la morfologia delle cellule epiteliali in pochi giorni di cultura, degeneri o si trasformano in fibroblasto-come le cellule.Poi, intorno al giorno 6 a 12, a contrasto di fase-brillante, tondo macrofago-come le cellule proliferano vigourouly sul foglio cellulare fibroblastica. Agitando dei flaconi cultura, i macrofagi sono prontamente sospese nel terreno di coltura, e il successivo trasferimento e l&#39;incubazione in piatti di plastica risultato in adesione selettiva dei macrofagi (Figura 3). Il macrofago-come le cellule possono essere raccolte fin dal giorno 8, e il numero ha raggiunto livelli massimi nei giorni 12 a 14. Più di 10 6 cellule possono essere raccolte da un pallone T75 cultura ripetutamente da 2 a intervalli di 3 al giorno per più di due settimane, consentendo un rendimento totale di cellule per bombola del 10 7 al T75 fiasco cultura (Figura 3). La purezza dei macrofagi isolati sono stati del 95 al 99%, come valutato da flowcytometry o immunocitochimica con ratto macrofagi anticorpi specifici, come ED-1 (Figura 4), ​​ED-3, OX-41 (Figura 4) e Iba1 9. Le cellule isolate mostrano caratteristiche tipiche dei macrofagi, come la fagocitosi attiva di perline polystylene (Figura 5), la risposta proliferativa a ricombinante GM-CSF, la secrezione di infiammatorio / citochine antinfiammatorie sulla stimolazione con LPS, e la formazione di cellule giganti multinucleate 9. <img src="/files/ftp_upload/2757/2757fig1.jpg" alt="Figura 1" /> Figura 1. Un sistema per l&#39;isolamento e la purificazione dei macrofagi come cellule miste colture primarie di cellule di fegato di ratto. <img src="/files/ftp_upload/2757/2757fig2.jpg" alt="Figura 2" /> Figura 2. Colture primarie di cellule di fegato di ratto e la proliferazione dei macrofagi come cellule. Freccia indica una cella multinucleare gigante. Barra di scala = 100 micron. Ristampato dalla Gazzetta di metodi immunologici, Vol.360, 47-55 (2010) 9 con il permesso di Elsevier. <img src="/files/ftp_upload/2757/2757fig3.jpg" alt="Figura 3" /> Figura 3. Isolamento selettivo di macrofagi come le cellule dalle scosse e il metodo di fissaggio. Le cellule sono state sospese nel terreno di coltura agitando i palloni, successivamente trasferito in non-tessuto piatti di plastica cultura grado, e incubate a 37 ° C. Già 10 minuti dopo la placcatura, macrofagi come le cellule attaccate alla superficie piatto, mentre altre cellule contaminanti fibroblastica è rimasta in sospeso. Dopo il risciacquo con PBS, una popolazione di macrofagi altamente purificato è stato ottenuto. Queste cellule guadagnato morfologia tipica dei macrofagi dopo 40 min di cultura e cellule mitotiche sono stati osservati frequentemente (frecce). Successive modifiche del numero delle cellule recuperato palloni in periodi di diversa cultura sono mostrati. I valori sono una media ± SD 3-5 fiaschi. Gli esperimenti sono stati ripetuti tre volte ed i dati sono indicate da simboli diversi. Barra di scala = 100 micron. Ristampato dalla Gazzetta di metodi immunologici, Vol.360, 47-55 (2010) 9 con il permesso di Elsevier. <img src="/files/ftp_upload/2757/2757fig4.jpg" alt="Figura 4" /> Figura 4. Colorazione immunocitochimica dei macrofagi come cellule con anticorpi monoclonali contro macrofagi di ratto. <img src="/files/ftp_upload/2757/2757fig5.jpg" alt="Figura 5" /> Figura 5. Fagocitosi di marcato con FITC microsfere da macrofagi come cellule.

<ol>
	<li>Crispe, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+liver+as+a+lymphoid+organ.">The liver as a lymphoid organ.</a> Annu. Rev. Immunol. 27, 147-163 (2009).</li><li>Roberts, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+Kupffer+cell+in+mediating+hepatic+toxicity+and+carcinogenesis.">Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis.</a> Toxicol. Sci. 96, 2-15 (2007).</li><li>Gao, B., Jeong, W. I., Tian, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver:+An+organ+with+predominant+innate+immunity.">Liver: An organ with predominant innate immunity.</a> Hepatology. 47, 729-736 (2008).</li><li>Janousek, J., Strmen, E., Gervais, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+murine+Kupffer+cells+by+centrifugal+elutriation.">Purification of murine Kupffer cells by centrifugal elutriation.</a> J. Immunol. Methods. 164, 109-117 (1993).</li><li>Olynyk, J. K., Clarke, S. L. <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+primary+culture+of+rat+Kupffer+cells.">Isolation and primary culture of rat Kupffer cells.</a> J. Gastroenterol. Hepatol. 13, 842-845 (1998).</li><li>Heuff, G., Meyer, S., Beelen, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+rat+and+human+Kupffer+cells+by+a+modified+enzymatic+assay.">Isolation of rat and human Kupffer cells by a modified enzymatic assay.</a> J. Immunol. Methods. 174, 61-65 (1994).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods Cell Biol. 13, 29-83 (1976).</li><li>Yoshioka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+expression+of+cytokine+mRNAs+by+lipopolysaccharide+in+bovine+Kupffer+cells.">Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells.</a> Vet. Immunol. Immunopathol. 58, 155-163 (1997).</li><li>Kitani, H., Takenouchi, T., Sato, M., Yoshioka, M., Yamanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+isolation+method+for+macrophage-like+cells+from+mixed+primary+cultures+of+adult+rat+liver+cells.">A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells.</a> J. Immunol. Methods. 360, 47-55 (2010).</li><li>Gorrell, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+fibrosis:+the+hepatocyte+revisited.">Liver fibrosis: the hepatocyte revisited.</a> Hepatology. 46, 1659-1661 (2007).</li></ol>

Tutti gli animali erano tenuti e gestiti nella struttura animale presso l&#39;Istituto Nazionale della Salute degli animali, secondo le linee guida e regolamenti stabiliti dal comitato dell&#39;istituzione per gli esperimenti sugli animali.

Qui riportiamo un metodo semplice ed efficace per ottenere macrofagi dalle culture miste primarie di cellule adulte fegato di ratto. Il nostro metodo si basa sul primo romanzo l&#39;attività proliferativa dei macrofagi nella cultura mista di cellule del fegato di ratto, e poi il successivo isolamento e la purificazione di queste cellule in base alle loro caratteristiche biologiche come macrofagi 9. Potrebbe essere possibile i macrofagi proliferato nella cultura misto primario delle cellule epatiche adulte potrebbero avere origine da cellule di Kupffer o correlati che contaminata nella frazione parenchimale epatociti. I macrofagi potrebbe avere risposto a specifici cambiamenti ambientali causati dalla trasformazione delle cellule parenchimali epatociti 10 e altre fibroblastica in colture miste primarie. In conclusione, il nostro metodo fornisce l&#39;isolamento di cellule macrofaghe in numero sufficiente e la purezza della cultura primario del fegato di ratto cellswithout utilizzando sofisticate apparecchiature e competenze tecniche. Inoltre, la procedura di isolamento può essere ripetuta con il pallone stessa cultura per più di due settimane. Questo metodo può essere applicabile ad altre specie di mammiferi.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> Soluzione di sodio pentobarbital </td><td> Kyoritsu Seiyaku </td><td> Somnopentyl iniezione </td><td></td></tr><tr><td> Sale di Hank soluzione equilibrata (HBSS) </td><td> Invitrogen </td><td> 14185-052 </td><td></td></tr><tr><td> Glicole etilenico tetracetico acido (EGTA) </td><td> Sigma-Aldrich </td><td> E-4378 </td><td></td></tr><tr><td> Collagenasi </td><td> Wako Pure Chemical Industries, Ltd. </td><td> 032-10534 </td><td> Dissociazione delle cellule di grado (Lotto controllo necessario) </td></tr><tr><td> Inibitori della tripsina </td><td> Sigma-Aldrich </td><td> T-9128 </td><td></td></tr><tr><td> Medio minimo indispensabile aquila (MEM) </td><td> Sigma-Aldrich </td><td> M-4655 </td><td></td></tr><tr><td> Dulbecco modificato Mezzo di Eagle (DMEM) </td><td> Sigma-Aldrich </td><td> D-6459 </td><td> Alta glucosio-tipo </td></tr><tr><td> Di siero fetale bovino (FCS) </td><td> Hyclone </td><td> SH30070.03 </td><td> Inattivato con il calore a 56 ° C per 30 min. (Lotto controllo necessario) </td></tr><tr><td> TrypLE espresso </td><td> Invitrogen </td><td> 12605 </td><td></td></tr><tr><td> Fosfato di soluzione salina tamponata Dulbecco (PBS) </td><td> Invitrogen </td><td> 14190 </td><td> Ca 2 +-Mg 2 + libero </td></tr><tr><td> β-mercaptoetanolo </td><td> Sigma-Aldrich </td><td> M-7522 </td><td> Magazzino: 100 mm di acqua distillata </td></tr><tr><td> Insulina </td><td> Sigma-Aldrich </td><td> I-5500 </td><td> Disponibile: 10 mg / ml in 0,1 N HCl </td></tr><tr><td> Penicillina / streptomicina </td><td> Invitrogen </td><td> 15070 </td><td></td></tr><tr><td> Cellula filtro </td><td> BD Biosciences </td><td> 352360 </td><td> Dimensioni di maglia: 100 micron </td></tr><tr><td> Tessuto cultura fiasco </td><td> Sumitomo Bakelite Co., Ltd. </td><td> MS-21250 </td><td> Superficie: 75 cm 2 </td></tr><tr><td> Non-tessuti piatti della cultura plastica </td><td> BD Biosciences </td><td> 351005 </td><td></td></tr><tr><td> Cell scraper </td><td> Corning Inc. </td><td> 3010 </td><td></td></tr><tr><td> Reciproca shaker </td><td> TAITEC </td><td> NR-1 </td><td></td></tr></tbody></table>

a simple and efficient method to isolate macrophages from mixed primary cultures of adult liver cells

Cellule di Kupffer del fegato sono specifici per macrofagi residenti e svolgono un ruolo importante nelle funzioni fisiologiche e patologiche del fegato 1-3. Anche se i metodi di isolamento dei macrofagi del fegato sono state ben descritte 4-6, la maggior parte di questi metodi richiedono apparecchiature sofisticate, come un elutriator centrifuga e competenze tecniche. Ecco, mettiamo a disposizione un nuovo metodo per ottenere i macrofagi del fegato in numero sufficiente e purezza da miste colture primarie di cellule adulte fegato di ratto, come schematicamente illustrato nella figura 1. Dopo la dissociazione delle cellule del fegato da due fasi metodo di perfusione 7,8, una frazione per lo più composto da epatociti parenchimale è preparato e seminato in fiasche T75 tessuto cultura con terreno di coltura costituito da DMEM e il 10% epatociti FCS.Parenchymal perdere la morfologia delle cellule epiteliali entro pochi giorni di cultura, degeneri o si trasformano in cellule fibroblasto-simili (Figura 2). Con il procedere della cultura, intorno al 6 ° giorno, contrasto di fase-brillante, tondo macrofagi, come le cellule a proliferare sul foglio delle cellule fibroblastica (Figura 2). La crescita del macrofago-come le cellule continuare e arrivare ai livelli massimi intorno al giorno 12, che copre strato di cellule sulla superficie del pallone. Agitando dei flaconi cultura, i macrofagi sono prontamente sospese nel terreno di coltura. Il successivo trasferimento e breve incubazione in plastica risultato piatti in adesione selettiva dei macrofagi (Figura 3), dove rimangono sospese come le altre cellule contaminanti. Dopo diversi lavaggi con PBS, i macrofagi allegato vengono raccolte. Più di 10 6 cellule possono essere raccolte più volte dallo stesso T75 fiasco colture di tessuti a intervalli di 2-3 giorni per più di due settimane (Figura 3). La purezza dei macrofagi isolati sono stati del 95 al 99%, valutata mediante citometria di flusso o immunocitochimica con ratto macrofagi anticorpi specifici (Figura 4). Le cellule isolate mostrano fagocitosi attiva di perline polystylene (Figura 5), ​​la risposta proliferativa a ricombinante GM-CSF, la secrezione di infiammatorio / citochine antinfiammatorie sulla stimolazione con LPS, e formazione di cellule giganti multinucleate 9. In conclusione, mettiamo a disposizione un metodo semplice ed efficace per ottenere i macrofagi del fegato in numero sufficiente e purezza senza attrezzature complesse e skills.This metodo potrebbe essere applicabile ad altre specie di mammiferi.

Watch this Scientific Journal Video about A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells at JoVE.com

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

Un nuovo metodo per ottenere i macrofagi da colture primarie di cellule di fegato di ratto è descritto. Questo metodo utilizza la proliferazione dei macrofagi nella cultura, seguita da agitazione di palloni cultura e la purificazione di attaccamento selettivo ai piatti di plastica. Questa tecnica fornisce in modo efficiente i macrofagi del fegato, senza attrezzature complesse e competenze.

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 ...

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JoVE Journal

Immunology and Infection

37.9K Views.  National Institute of Agrobiological Sciences, Tsukuba, Japan. Un nuovo metodo per ottenere i macrofagi da colture primarie di cellule di fegato di ratto è descritto. Questo metodo utilizza la proliferazione dei macrofagi nella cultura, seguita da agitazione di palloni cultura e la purificazione di attaccamento selettivo ai piatti di plastica. Questa tecnica fornisce in modo efficiente i macrofagi del fegato, senza attrezzature complesse e competenze.

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

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 ...

Ricerca

Immunologia e infezioni

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 ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

Un metodo semplice ed efficace per rilevare l&#39;attivazione del Fattore Nucleare in neutrofili umani mediante citometria a flusso

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Trasfezione altamente efficiente di umani THP-1 da macrofagi Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Un metodo del rendimento efficiente e di alta per l&#39;isolamento di mouse dendritiche sottopopolazioni cellulari

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Un approccio semplice ed efficiente per costruire Mutant vaccinia vettori di virus

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Generazione e identificazione di GM-CSF Derivato alveolo-come macrofagi e dendritiche cellule da midollo osseo di topo

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

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 ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Un modo non invasivo per isolare e fenotipo le cellule della Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

Un metodo semplice ed efficace per stabilire NK e linee a cellula T da pazienti con infezione attiva cronica del Virus di Epstein - Barr

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...