Questo lavoro è stato parzialmente sostenuto da una sovvenzione di Fund-A-Fellow della Fondazione Myotonic Dystrophy (MDF-FF-2014-0013) e dello Scienziato per la Sviluppo dell&#39;American Heart Association (16SDG29320001).

I topi transgenici Wildtype e Nestin-GFP sono stati alloggiati nella struttura animale presso l&#39;Università del Minnesota. Tutte le procedure sperimentali sono state approvate dal Comitato istituzionale per la cura e l&#39;uso degli animali presso l&#39;Università del Minnesota e sono stati conformi alla Guida NIH per la cura e l&#39;uso degli animali da laboratorio. 1. Dissezione del muscolo e isolamento a cellule singole <ol><li> Eutanizzare i topi adulti (6-10 settimane, sia maschi che femmine) con il tribromoetanolo (250 mg / kg, ip) e sterilizzare la loro pelle dell&#39;addome con il 70% di etanolo. NOTA: il Tribromoetanolo è stato usato qui invece che la ketamina per l&#39;anestesia / eutanasia, in quanto la chetamina è nota per interagire con i recettori NMDA, che potrebbero avere un impatto sullo studio. </li><li> Posizionare i topi in posizione supina e utilizzare un bisturi per eseguire un&#39;incisione orizzontale sulla pelle addominale. Scorri la pelle manualmente, tirando in direzioni opposte per esporre i muscoli posteriori. </li><li> RaccolteI muscoli di entrambi i muscoli posteriori usando pinze e forbici. Conservarli in salina sterilizzata tamponata fosfato (PBS) integrata con 1% di penicillina-streptomicina (P / S) sul ghiaccio. </li><li> Lavare i muscoli dell&#39;indietro dissected in PBS ghiacciato-freddo completato con 1% P / S due volte e trasferirli in una piastra sterile da 10 cm. </li><li> Separare con cautela i nervi, i vasi sanguigni e il tessuto connettivo dai muscoli usando le pinze e le forbici sotto un microscopio di dissezione ad ingrandimento 2X. </li><li> Tagliare finemente e tagliare i muscoli in piccoli pezzi (1-2 mm 3 ) usando le forbici sterili e le lame. Se necessario, aggiungere una piccola quantità di Dulbecco&#39;s Modified Eagle Medium (DMEM) per assicurare che i muscoli non venissero asciugati. </li><li> Determinare meccanicamente il tessuto pipettando su e giù attraverso una pipetta serologica da 10 ml 10 volte. </li><li> Aggiungere la soluzione di digestione appena fatta (DMEM integrata con 0,2% di collagene tipo 2) alla miscela. Incubare a 37 ° C per 2 ore con gentlAgitazione a 35 giri / min. </li><li> Tritare usando un ago da 18 G per omogeneizzare la miscela. Quindi, centrifugare a 500 xg per 5 min. Scartare il surnatante e quindi risospendere il pellet nello 0,25% di tripto / EDTA. Incubare a 37 ° C per 10 min. Ripetere la fase di centrifugazione ancora due volte. </li><li> Aggiungere 10 ml di DMEM completato con 20% di siero fetale fetale (FBS) alla soluzione e centrifugare a 500 xg per 5 min. Scartare il surnatante e risospendere il pellet nel tampone di lisi di globuli rossi (155 mM NH 4 Cl, 10 mM KHCO3 e 0,1 mM EDTA). </li><li> Centrifugare a 500 xg per 5 min. Scartare il surnatante e risospendere il pellet nel tampone di smistamento (20 mM HEPES, pH 7,0, 1 mM EDTA e 1% BSA in PBS Ca / Mg 2+ , pH 7,0). Filtrare la miscela attraverso un filtro da 40 μm per ottenere una sospensione a singola cellula. </li><li> Centrifugare a 500 xg per 5 min. Scartare il supernatante e risospendere il pellet in 1 mL di tampone di smistamento. </li><li> Conti ilNumero di cellule con un emocitometro e diluire la sospensione a singola cellula a 5 x 10 6 / mL nel tampone di smistamento. </li></ol> 2. Colorazione e ordinamento delle cellule <ol><li> Preparare i controlli e il campione come descritto nella tabella 1 . Macellare la sospensione a singola cellula con i rispettivi anticorpi sul ghiaccio per 30 minuti, come descritto nella tabella 1 . </li><li> Centrifugare a 500 xg per 5 minuti e lavare i pellet due volte con il tampone di smistamento. </li><li> Aggiungere DAPI alle soluzioni a cellule singole, come indicato nella tabella 1. Utilizzare 5 μg / mL DAPI (concentrazione finale) per il controllo monocromatico DAPI e 1 μg / mL DAPI per il controllo PDGFRβ-PE-FMO e il campione. Tenere tutti i tubi sul ghiaccio durante l&#39;esperimento. </li><li> Accendere il destinatario e il software. Eseguire la scansione e inserire un chip di classificazione da 100 μm quando richiesto. </li><li> Eseguire l&#39;impostazione automatica ( ad esempio l&#39; allineamento del chip, la calibrazione della gocciolina, la calibrazione dei flussi laterali e il ritardo di ordinamento calibratiOn) caricando le perline di impostazione automatica quando richiesto. </li><li> Una volta completata l&#39;installazione automatica, vai alla scheda &quot;Esperimento&quot;, fai clic su &quot;Nuovo&quot; e seleziona &quot;Modello vuoto&quot; da &quot;Modelli pubblici&quot;. </li><li> Sotto &quot;Impostazioni di misura&quot; immettere &quot;DAPI&quot; per &quot;FL1&quot;, &quot;Nestin-GFP&quot; per &quot;FL2&quot; e &quot;PDGFRβ&quot; per &quot;FL3.&quot; Deselezionare le caselle per &quot;FL4&quot; - &quot;FL6&quot;. </li><li> Controlla le caselle per attivare i laser 405, 488 e 561 e fai clic su &quot;Crea nuovo esperimento&quot;. </li><li> Selezionare l&#39;opzione &quot;Start Compensation Wizard&quot; e seguire le istruzioni software &quot;Compensation Wizard&quot; per impostare la compensazione. <ol><li> Carica il controllo non conservato e fai clic su &quot;Avvia&quot;. Fare clic su &quot;Detector &amp; Threshold Settings&quot; e regolare il guadagno del sensore dei sensori FSC e BSC per mettere la popolazione sulla bilancia. </li><li> Anno DominiSolo i livelli di guadagno dei flussi di fluorescenza FL1-FL3 per posizionare le popolazioni negative sul lato sinistro degli istogrammi. Fare clic sul pulsante &quot;Registra&quot; per registrare i dati. </li><li> Caricare controlli monocromatici uno a uno quando richiesto. Fai clic su &quot;Avviare e registrare&quot; per registrare i dati. Regolare i cancelli per le popolazioni positive sugli istogrammi. Fai clic su &quot;Avanti&quot;. </li><li> Vai a &quot;Calcola Matrice&quot; nella scheda &quot;Compensi&quot; e fai clic su &quot;Calcola&quot; nel pannello &quot;Calcola impostazioni di compensazione&quot; per eseguire la compensazione. Fai clic su &quot;Fine&quot; per uscire dalla &quot;Creazione guidata remunerazione&quot;. </li></ol></li><li> Caricare il controllo PDGFRβ-PE-FMO e fare clic su &quot;Start&quot;. Disegna una porta poligonale (Porta A) attorno alle celle di interesse sotto il grafico &quot;Tutti gli eventi&quot;. </li><li> Fare doppio clic all&#39;interno della porta A per creare una trama figlia. Cambiare l&#39;asse Y su DAPI e disegnare una porta poligonale (Porta B) attorno alle celle viventi (DAPI low ). Fare doppio clic su iNside Gate B per creare una trama figlia. Cambiare l&#39;asse X su FSC-H e l&#39;asse Y a FSC-W e disegnare una porta poligonale (Porta C) attorno alle singole per eliminare i dublettini. </li><li> Fare doppio clic all&#39;interno della porta C per creare una trama figlia. Cambiare l&#39;asse X a Nestin-GFP e l&#39;asse Y a PDGFRβ-PE. Fare clic sul pulsante &quot;Registra&quot; per registrare i dati. </li><li> Caricare il campione e ripetere i passaggi 2.11-2.12. Dopo la registrazione, fai clic su &quot;Pausa&quot; per conservare il campione. </li><li> Definire i limiti di gating per le cellule PDGFRβ + e Nestin-GFP + basate sul controllo PDGFRβ-PE-FMO. Disegnare le porte per le popolazioni di PDGFRβ + Nestin-GFP e PDGFRβ + Nestin-GFP + . </li><li> Sotto &quot;Metodo di ordinamento&quot;, selezionare &quot;Tubi a 2 vie&quot; e assegnare le celle &quot;PDGFRβ + Nestin-GFP&quot; e &quot;PDGFRβ + Nestin-GFP + &quot; ai tubi di raccolta destro e sinistrospettivamente. Montare i tubi di raccolta 15 ml di riempimento con tampone di riempimento nella fase di raccolta e fare clic sul pulsante &quot;Load Collection&quot;. </li><li> Fai clic sul pulsante &quot;Riprendi&quot; per mantenere il campione in esecuzione. Fare clic su &quot;Start Sort&quot; per raccogliere le cellule PDGFRβ + Nestin-GFP (periciti di tipo I) e PDGFRβ + Nestin-GFP + (periciti di tipo II). </li></ol> 3. Analisi post-ordinamento <ol><li> Centrifugare le cellule ordinate a 500 xg per 5 minuti, risospendere il pellet in 1 mL di mezzo di pericce (vedere tabella dei materiali ) e contare la densità cellulare usando un emocitometro. </li><li> Periciti di tipo I e II di tipo su copertine rivestite con poli-D-lisina (PDL) a ~ 1 x 10 4 cellule / cm 2 . Crescere in media pericta per 3 giorni a 37 ° C con 5% CO 2 . </li><li> Il giorno 3, esaminare la morfologia pericita (sotto il contrasto di fase) e l&#39;espressione endogena di Nestin-GFP utilizzando un micro fluorescenteCampo di applicazione (laser di eccitazione: 488 nm, filtro di eccitazione: 470/40 nm e filtro di emissione: 515/30 nm). Prendere le immagini sotto un obiettivo 20X (0,45 NA). </li><li> Sostituire il mezzo di pericite con adipogenic (mezzo di base basale MSC mouse + supplemento stimolatorio adipogenico) e myogenic (DMEM + 2% cavallo di cavallo) per iniziare rispettivamente la differenziazione adipogenica e miogenica, come descritto in precedenza 20 . Cambiare il supporto ogni 2-3 giorni. </li><li> Fissare le cellule il giorno 17 (14 giorni dopo la differenziazione adipogenica / miogenica) in 4% di paraformaldeide (PFA) per 20 minuti a temperatura ambiente. NOTA: Attenzione, PFA è un cancerogeno. </li><li> Eseguire l&#39;immunocitochimica contro la perilipina (marcatore di adipociti) e la S-myosina (marcatore miootubo / miofibre maturo), come descritto nelle pubblicazioni precedenti 19 , 20 . <ol><li> Lavare le cellule fisse 3 volte in PBS per 10 minuti a temperatura ambiente. </li><li> Aggiungere buffer di blocco (PBSCompleta di siero di asina al 5%, 3% BSA e 0,3% Triton X-100) e incubare a temperatura ambiente per 1 ora. </li><li> Incubare le cellule con anticorpi anti-perilipina (2 μg / mL) e / o anti-S-myosina (2 μg / mL) a 4 ° C per una notte. </li><li> Lavare le cellule 3 volte in PBS per 10 minuti a temperatura ambiente. </li><li> Incubare le cellule con anticorpi anti-coniglio di Alexa 555 (4 μg / mL) e / o Alexa555 anti-mouse (4 μg / mL) a temperatura ambiente per 1 ora. </li><li> Lavare le cellule 3 volte in PBS per 10 minuti a temperatura ambiente. </li><li> Montare le cellule immunocoste con supporto di supporto contenente DAPI (vedere la tabella dei materiali). Esaminare la perilipina e l&#39;espressione di S-myosina usando un microscopio fluorescente (laser di eccitazione: 543 nm, eccitazione 540/45 nm e emissione di 600/50 nm) e fotografare con un obiettivo 40X (0,60 NA). </li></ol></li></ol>

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Periciti sono cellule perivascolari multipotenti 22 , 23 situate sulla superficie abluminale dei capillari 21 , 26 . Nei muscoli scheletrici, i periciti sono in grado di differenziarsi lungo i percorsi adipogenici e / o miogenici 19 , 20 , 24 . Recenti studi hanno rivelato due sottopopolazioni di periciti, con d...

La distrofia muscolare è un disturbo muscolare-degenerativo che non ha finora trattamenti efficaci. Lo sviluppo di terapie che promuovono la rigenerazione dei tessuti è sempre stato di grande interesse. La rigenerazione tissutale e la riparazione dopo danni dipendono da cellule staminali residui / cellule progenitrici 1 . Le cellule satellitari sono impegnate da cellule precursori myogeniche che contribuiscono alla rigenerazione muscolare 2 , 3 , 4 , 5 , 6 , 7 . Tuttavia, il loro utilizzo clinico è ostacolato dalla loro limitata migrazione e dal basso tasso di sopravvivenza dopo l&#39;iniezione, nonché dalla loro diminuita capacità di differenziazione dopo l&#39;amplificazione in vitro 8 , 9 , 10 , 11 . Oltre ai satellitiLe cellule ei muscoli scheletrici contengono anche molte altre popolazioni cellulari con potenzialità myogenica 12 , 13 , 14 , 15 , 16 come le cellule interstiziali posizionali di fattore di crescita del recettore beta-beta (PDGFRβ) derivato dalle piastrine. Ci sono prove che dimostrano che le cellule PDGFRβ + derivate dal muscolo sono in grado di differenziarsi in cellule miogeniche e migliorare la patologia muscolare e la funzione 14 , 17 , 18 , 19 , 20 . PDGFRβ prevalentemente etichetta periciti 21 , che sono cellule perivascolari con pluripotenza 22 , 23 . Oltre al PDGFRβ, molti altri marcatori, tra cui Neuron-Glial 2 (NG2) e CD146, sono utilizzati anche per iDentify pericytes 21 . Va comunque osservato che nessuno di questi marcatori è specifico per cicli 21 . Recenti studi hanno rivelato due sottotipi di periciti muscolari, chiamati tipo I e tipo II, che esprimono diversi marcatori molecolari e svolgono funzioni distinte 19 , 24 , 25 . Biochimicamente, le pericelle di tipo I sono NG2 + Nestin, mentre le pericelle di tipo II sono NG2 + Nestin + 19 , 24 . Funzionariamente, i periciti di tipo I possono subire differenziazione adipogenica, contribuendo all&#39;accumulo di grassi e / o alla fibrosi, mentre i periciti di tipo II possono differenziarsi lungo il percorso miocogenico, contribuendo alla rigenerazione muscolare 19 , 24 , 25 . Questi risultati dimostrano che: (1) i periciti di tipo I possono bE destinato al trattamento di disturbi degenerativi grassi / fibrosi, e (2) le pericie di tipo II hanno un grande potenziale terapeutico per la distrofia muscolare. Ulteriori indagini e caratterizzazione di queste popolazioni richiedono un protocollo di isolamento che consente la separazione dei periciti di tipo I e tipo II ad un alto livello di purezza. Attualmente, l&#39;isolamento delle sottopopolazioni pericite si basa sui topi doppi transgenici NG2-DsRed e Nestin-GFP 19 , 24 . La disponibilità di topi NG2-DsRed e la qualità della maggior parte degli anticorpi NG2 limitano l&#39;uso diffuso di questo metodo. Dato che tutti i periciti NG2 + esprimono PDGFRβ nei muscoli scheletrici 19 , 20 , 24 , ipotizziamo che NG2 può essere sostituito da PDGFRβ per l&#39;isolamento delle periciti e delle loro subpopulazioni. Questo lavoro descrive un protocollo basato su FACS cheUtilizza la colorazione PDGFRβ e il segnale Nestin-GFP. Questo metodo è meno impegnativo per gli investigatori perché: (1) non richiede lo sfondo NG2-DsRed e (2) utilizza gli anticorpi PDGFRβ commercialmente disponibili, ben caratterizzati. Inoltre, consente l&#39;isolamento simultaneo di periciti di tipo I e di tipo II ad alta purezza, permettendo di approfondire la biologia e il potenziale terapeutico di queste sottopopolazioni pericite. A seguito della purificazione, queste cellule possono essere coltivate in coltura e le loro morfologie possono essere visualizzate. Questo lavoro mostra anche che i periciti di tipo I e II tipo isolati utilizzando questo metodo, come quelli purificati da topi doppi transgenici, sono rispettivamente adipogenici e miogenici.

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			<td height="22" style="height:22px;width:311px;">Fetal Bovine Serum</td>
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			<td height="22" style="height:22px;width:311px;">Alexa 555-anti-rabbit antibody&#160;</td>
			<td style="width:173px;">ThermoFisher Scientific</td>
			<td style="width:133px;">A-31572</td>
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			<td height="22" style="height:22px;width:311px;">Alexa 555-anti-mouse antibody</td>
			<td style="width:173px;">ThermoFisher Scientific</td>
			<td style="width:133px;">A-31570</td>
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			<td height="22" style="height:22px;width:311px;">Mounting Medium with DAPI</td>
			<td style="width:173px;">Vector Laboratories</td>
			<td style="width:133px;">H-1200</td>
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			<td height="22" style="height:22px;width:311px;">DAPI</td>
			<td style="width:173px;">ThermoFisher Scientific</td>
			<td style="width:133px;">D1306</td>
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			<td style="width:133px;">A2058</td>
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			<td style="width:173px;">Fisher Scientific</td>
			<td style="width:133px;">A661</td>
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			<td height="26" style="height:26px;width:311px;">KHCO3</td>
			<td style="width:173px;">Fisher Scientific</td>
			<td style="width:133px;">P184</td>
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			<td height="22" style="height:22px;width:311px;">18G Needles</td>
			<td style="width:173px;">BD</td>
			<td style="width:133px;">305196</td>
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			<td height="22" style="height:22px;width:311px;">10ml Serological Pipette</td>
			<td style="width:173px;">BD</td>
			<td style="width:133px;">357551</td>
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isolation of type i and type ii pericytes from mouse skeletal muscles

Periciti sono cellule multipotenti perivascolari che mostrano eterogeneità in organi diversi o addirittura all&#39;interno dello stesso tessuto. Nei muscoli scheletrici esistono almeno due sottopopolazioni pericite (chiamate tipo I e tipo II) che esprimono differenti marcatori molecolari e hanno differenti capacità di differenziazione. Utilizzando i topi a doppio-transgenici NG2-DsRed e Nestin-GFP, le pericelle di tipo I (NG2-DsRed + Nestin-GFP-) e di tipo II (NG2-DsRed + Nestin-GFP + ) sono state isolate con successo. Tuttavia, la disponibilità di questi topi doppi transgenici impedisce l&#39;uso diffuso di questo metodo di purificazione. Questo lavoro descrive un protocollo alternativo che consente l&#39;isolamento facile e simultaneo di periciti tipo I e II di tipo muscolare scheletrico. Questo protocollo utilizza la tecnica di selezione delle cellule attivata con fluorescenza (FACS) e mira a PDGFRβ, piuttosto che a NG2, insieme al segnale Nestin-GFP. Dopo l&#39;isolamento, digitare I e tyPe periciti mostrano morfologie distinte. Inoltre, le pericie di tipo I e tipo II isolate con questo nuovo metodo, come quelle isolate dai topi doppi transgenici, sono rispettivamente adipogenici e miogenici. Questi risultati suggeriscono che questo protocollo può essere utilizzato per isolare le sottopopolazioni pericite dai muscoli scheletrici e, probabilmente, da altri tessuti.

I parametri FACS, compresa l&#39;intensità laser e la compensazione dei canali, vengono corretti in base ai risultati di controlli non controllati e controlli monocromatici. Il controllo PDGFRβ-PE-FMO viene utilizzato per impostare il gating per la popolazione di PDGFRβ-PE + ( Figura 1A ). Tra le cellule PDGFRβ-PE, due popolazioni che rappresentano le cellule Nestin-GFP + e Nestin-GFP sono chiaramente separate ( <strong class="xfig"...

Watch this Scientific Journal Video about Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles at JoVE.com

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Questo lavoro descrive un protocollo basato su FACS che consente l&#39;isolamento facile e simultaneo di periciti tipo I e II di tipo muscolare scheletrico.

Isolamento delle pericelle di tipo I e II di muscoli scheletrici del mouse

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

isolation-of-type-i-and-type-ii-pericytes-from-mouse-skeletal-muscles

Research

JoVE Journal

Developmental Biology

9.1K Views.  University of Minnesota.  Questo lavoro descrive un protocollo basato su FACS che consente l'isolamento facile e simultaneo di periciti tipo I e II di tipo muscolare scheletrico. 

Video: Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

Isolamento e quantitativa immunocitochimica caratterizzazione di cellule miogeniche primarie e fibroblasti da muscolo scheletrico umano

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Ricerca

Biologia dello sviluppo

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Isolamento e caratterizzazione di cellule satelliti da muscoli Rat Capo Branchiomeric

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Utilizzo di mitocondri isolati da minime quantità di muscolo scheletrico del mouse per High micropiastre rendimento respiratorie Misure

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Efficace isolamento di isole funzionali da neonatali mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

L&#39;isolamento e arricchimento di fegato progenitrici sottoinsiemi identificati da un marcatore combinazione romanzo di superficie

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

Generazione di standardizzato e riproducibile del Forebrain-tipo Organoids cerebrale da umani cellule staminali pluripotenti indotte

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

Dissezione della drosofila Pupal Retina per immunoistochimica, analisi occidentale e isolamento del RNA

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

Isolamento, cultura, caratterizzazione e differenziazione delle cellule del progenitore muscolare umano dalla procedura di biopsia muscolare scheletrica

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Isolamento e differenziazione dei mioblasti primari dagli esopanti muscolari scheletrici del topo

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

Tradurre la purificazione dell'affinità del ribosoma (TRAP) per studiare lo sviluppo della radice di Arabidopsis thaliana su una scala specifica del tipo di cella

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...