Ringraziamo i membri della divisione di scienza del genoma e laboratorio per sistemi di biologia e medicina, RCAST, The University of Tokyo. Ringraziamo in particolare Dr. H. Aburatani, Dr. T. Kodama, (LSBM, RCAST, l'Università di Tokyo), Dr. K. Tomizuka, Dr. T. Yoshida e Dr. A. Kunisato (Kyowa Hakko Kirin Co., Ltd.) per utili discussioni e supporto. Quest'opera è stata in parte sostenuta da sovvenzione per scienziato (A) (26710005, T.O.), del giovane sovvenzione per la ricerca scientifica in settori innovativi (26116711 e 16 H 01567, T.O.) e sovvenzione per sfidare Ricerca esplorativa (16K 14605, T.O.) dal Ministero della pubblica istruzione, cultura, sport, scienza e tecnologia del Giappone, la Fondazione di scienza di Takeda (T.O.), la Fondazione di Kobayashi della ricerca sul cancro (T.O.) e il progetto per la ricerca sul cancro e Evoluzione terapeutica (P-creare) e la ricerca pratica per la gestione innovativa cancro dal Giappone Agenzia per la ricerca medica e sviluppo, AMED (T.O.).

1. preparazione del terreno di coltura acida
<ol><li>
Preparazione del controllo (pH 7,4) e terreno di coltura di basso pH (pH 6,8)
	<ol>
<li>Sciogliere 4,75 g di polvere DMEM senza L-Glutammina in 500 mL di acqua.</li>
		<li>Preparare 0,33 M NaHCO3 soluzione in una bottiglia a tenuta di pressione. Attenzione: È importante utilizzare una bottiglia di tenuta pressione poiché NaHCO3 emette CO2 gas quando riscaldato e decomposizione termica.</li>
		<li>Autoclave il mezzo e soluzione. Consentire i buffer raffreddare a 25 ° C.</li>
		<li>Aggiungere 5 mL di 200 mM L-Glutammina, 5 mL di penicillina-streptomicina e 50 mL di siero fetale bovino a 500 mL di DMEM senza bicarbonato.</li>
		<li>Per mezzo di controllo (pH 7.4), aggiungere 2,4 mL di soluzione di 0,33 M NaHCO3 per 100 mL di DMEM senza bicarbonato. Per mezzo di basso pH (pH 6,8), aggiungere 0,6 mL di soluzione di 0,33 M NaHCO3 per 100 mL di DMEM senza bicarbonato.</li>
		<li>Controllare il pH medio dopo 24 h di incubazione a 37 ° C inferiore al 5% di CO2. Nota: La quantità di soluzione di NaHCO3 deve essere regolato a seconda del pH medio, che possa differire a seconda del contenuto di CO2 del incubatrice e pressione dell'aria. Idealmente, per la prima volta, è meglio misurare il pH medio con varie concentrazioni di bicarbonato per determinare la quantità appropriata di NaHCO3 soluzione per aggiungere a DMEM.<ol>
<li>Raccogliere il terreno di coltura immediatamente dopo aver tolto la piastra di coltura dell'incubatrice di CO2 e misurare il pH utilizzando un pH-metro. Mantenere la temperatura media a 37 ° C con un bagno d'acqua se necessario. Misurare il pH medio tre volte in modo indipendente. Nota: NaHCO3 soluzione deve essere conservata a 4 ° C e ogni mass media deve essere preparata. Prodotti disponibili in commercio che possono essere utilizzati in questo studio sono elencati in Tabella materiali.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Preparazione terreno di coltura acida indotta da lattato o indotta da HCl
	<ol>
<li>Aggiungere 100 mL di terreno di controllo (preparata al punto 1.1.5) 74 µ l di soluzione del lattato o 125 µ l di 1 M HCl.</li>
		<li>Controllare il pH medio dopo 24 h di incubazione a 37 ° C inferiore al 5% di CO2. Nota: La quantità di lattato o soluzione di HCl deve anche essere regolata secondo il pH medio, che possa differire a seconda del contenuto di CO2 nel incubatrice e pressione dell'aria. Idealmente, è meglio misurare il pH medio con seriale lattato o concentrazioni di HCl per determinare la quantità appropriata per la regolazione del pH desiderato del mezzo.</li>
	</ol>
</li>
</ol>2. raccolta e trattamento di cellule
<ol><li>Iniziare a coltivare le cellule a 37 ° C, inferiore al 5% di CO2 almeno 1 settimana prima di eseguire esperimenti. Nota: Le linee cellulari utilizzate in questo studio sono elencate in Tabella materiali.</li>
	<li>Cellule di passaggio quando raggiungono confluency di 70-90%. Non permettono alle cellule di diventare confluenti. Modificare il mezzo ogni 2-3 giorni durante la coltivazione quotidiana.</li>
	<li>Lavare le cellule aggiungendo 5 mL di PBS. Aspirare il PBS e aggiungere enzima distaccante quali 1 mL di tripsina.</li>
	<li>Incubare a 37 ° C fino a quando le cellule vengono arrotondate e iniziano a staccare.</li>
	<li>Aggiungere 5 mL di terreno di coltura le cellule indipendenti e disattivare degli enzimi. Trasferire la sospensione cellulare in una provetta da 15 mL e centrifugare per 3 min a 300 x g. aspirare il supernatante e risospendere le cellule con terreno di coltura.</li>
	<li>Contare le cellule vitali utilizzando un emocitometro e blu di Trypan.</li>
	<li>Seme 5,0 x 105 cellule/pozzetto di un piatto di 10 cm in 10 mL di terreno. Incubare le cellule per 24 h a 37 ° C inferiore al 5% di CO2.</li>
	<li>Aspirare il terreno di coltura, lavare le cellule con 5 mL di PBS e aggiungere 10 mL di terreno di coltura acido preparato nella sezione 1. Incubare le cellule per 24 h a 37 ° C inferiore al 5% di CO2.</li>
</ol>3. RNA isolamento
<ol><li>Aspirare il terreno di coltura e lavare le cellule con 5 mL di PBS. Frantumare le cellule con l'aggiunta del reagente di estrazione di RNA (Vedi Tabelle materiali).</li>
	<li>Raschiare il piatto brevemente, quindi rimuovere il reagente di estrazione di RNA con una pipetta e depositare il RNA estrazione reagente/lysate delle cellule in una provetta da 1,5 mL. Lasciare a temperatura ambiente per 5 min.</li>
	<li>Aggiungere 250 cloroformio µ l e agitare la provetta per lasciare circa 15 s. a temperatura ambiente per 5 min e centrifugare a ≥ 8, 000 x g per 5 min.</li>
	<li>Rimuovere con cautela la fase acquosa utilizzando una pipetta. Lasciare dietro alcuni della fase acquosa. Posto in un'altra provetta da 1,5 mL.</li>
	<li>Aggiungere 550 µ l isopropanolo alla fase acquosa e mescolare delicatamente. Lasciare a temperatura ambiente per 5 min. Centrifugare a massima velocità (≥ 20, 000 x g) per 20 min a 4 ° C.</li>
	<li>Versare fuori l'isopropanolo e aggiungere 1 mL di etanolo al 75% in DEPC acqua trattata. Mescolare delicatamente. Centrifugare a 8.000 x g per 5 min.</li>
	<li>Pipettare fuori l'etanolo e lasciare che asciugare all'aria il pellet.</li>
	<li>Aggiungere che circa 15-25 µ l (a seconda del rendimento) di DEPC acqua trattata per la pallina del RNA. Mescolare accuratamente.</li>
	<li>Misurare la concentrazione di RNA isolato misurando l'assorbanza a 260 nm utilizzando uno spettrofotometro.</li>
</ol>4. sintesi del cDNA
<ol><li>Combinare i seguenti componenti in un tubo di reazione di PCR: fino a 5 µ g RNA totale, 1 µ l Oligo dT (50 µM) o Primer Mix (60 µM), 1 µ l 10 mM dNTP e acqua priva di nucleasi per un volume totale di 14 µ l.</li>
	<li>Mescolare e centrifugare brevemente i componenti utilizzando una centrifuga da banco piccolo (~ 5 s). Denaturare il campione primer di RNA per 5 min a 65 ° C. Girare brevemente (~ 5 s) utilizzando un piccolo benchtop centrifugare e mettere il campione immediatamente in ghiaccio per almeno 1 min.</li>
	<li>Aggiungere i seguenti componenti: 4 µ l 5 x buffer della trascrizione inversa, 1 µ l della trascrittasi inversa (200 U / µ l), 1 µ l della RNAsi inibitore (40 U / µ l). Nota: Esempi di kit commercialmente disponibili sono mostrati in Tabella materiali
</li>
	<li>Richiudere la provetta, mescolare e poi Centrifugare brevemente il contenuto utilizzando una centrifuga da banco piccolo (~ 5 s). Incubare la miscela di reazione combinata a 50-55 ° C per 10 min.</li>
	<li>Inattivare la reazione incubando a 80 ° C per 10 min.</li>
</ol>5. misurazione dell'espressione genica acido-sensibli a reagire mediante PCR in tempo reale
<ol><li>Combinare i seguenti componenti in una piastra 384 pozzetti di reazione: 1 µ l modello cDNA, 0,75 µ l 10 X N',N'-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine, 0,3 µM 50 µ l in avanti Primer, 0,3 µ l 50 µM reverse primer, 1 µ l Taq polimerasi, 2,5 µ l 10 x buffer della polimerasi di Taq, 2,5 µ l 20 mM dNTP, 16.65 µ l acqua. Nota: Esempio di kit commercialmente disponibili vengono visualizzati nella Tabella materiali. Nella tabella 1viene visualizzato un elenco degli iniettori. Una piastra a 96 pozzetti di reazione può essere utilizzata anche in questo passaggio. Altre sonde (ad es., sonda Taqman) possono anche essere utilizzate.</li>
	<li>Impostare l'esperimento e il seguente programma PCR su un sistema di PCR in tempo reale: 50 ° C per 2 min, 1 ciclo; 95 ° C per 10 min, 1 ciclo; 95 ° C per 15 s e 60 ° C per 1 min, 40 cicli; 95 ° C per 15 s, 1 ciclo; 60 ° C per 15 s, 1 ciclo; 95 ° C per 15 s, 1 ciclo.</li>
	<li>Posizionare la piastra nello strumento qPCR. Avviare il programma. Nota: Upregulation dei geni reattivo pH MSMO1, INSIG1e IDI1 può essere misurata dai livelli della proteina.</li>
</ol>

<ol>
	<li>Cairns, R. A., Harris, I. S., Mak, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cancer+cell+metabolism.">Regulation of cancer cell metabolism.</a> Nature reviews. Cancer. 11 (2), 85-95 (2011).</li><li>Chang, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Competition+in+the+Tumor+Microenvironment+Is+a+Driver+of+Cancer+Progression.">Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression.</a> Cell. 162 (6), 1229-1241 (2015).</li><li>Hsu, P. P., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+metabolism:+Warburg+and+beyond.">Cancer cell metabolism: Warburg and beyond.</a> Cell. 134 (5), 703-707 (2008).</li><li>Gerweck, L. E., Seetharaman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+pH+gradient+in+tumor+versus+normal+tissue:+potential+exploitation+for+the+treatment+of+cancer.">Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer.</a> Cancer research. 56 (6), 1194-1198 (1996).</li><li>Webb, B. A., Chimenti, M., Jacobson, M. P., Barber, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulated+pH:+a+perfect+storm+for+cancer+progression.">Dysregulated pH: a perfect storm for cancer progression.</a> Nature Rev. Cancer. 11 (9), 671-677 (2011).</li><li>Gatenby, R. A., Gillies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+cancers+have+high+aerobic+glycolysis?.">Why do cancers have high aerobic glycolysis?.</a> Nature reviews. Cancer. 4 (11), 891-899 (2004).</li><li>McBrian, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+regulates+intracellular+pH.">Histone acetylation regulates intracellular pH.</a> Mol Cell. 49 (2), 310-321 (2013).</li><li>Damaghi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+acidosis+in+the+tumour+microenvironment+selects+for+overexpression+of+LAMP2+in+the+plasma+membrane.">Chronic acidosis in the tumour microenvironment selects for overexpression of LAMP2 in the plasma membrane.</a> Nat Commun. 6, 8752 (2015).</li><li>Corbet, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acidosis+Drives+the+Reprogramming+of+Fatty+Acid+Metabolism+in+Cancer+Cells+through+Changes+in+Mitochondrial+and+Histone+Acetylation.">Acidosis Drives the Reprogramming of Fatty Acid Metabolism in Cancer Cells through Changes in Mitochondrial and Histone Acetylation.</a> Cell Metab. 24 (2), 311-323 (2016).</li><li>Chen, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+analysis+of+lactic+acidosis+and+acidosis+response+in+human+cancers.">The genomic analysis of lactic acidosis and acidosis response in human cancers.</a> PLoS genetics. 4 (12), e1000293 (2008).</li><li>Kondo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Acidic+pH+Activates+the+Sterol+Regulatory+Element-Binding+Protein+2+to+Promote+Tumor+Progression.">Extracellular Acidic pH Activates the Sterol Regulatory Element-Binding Protein 2 to Promote Tumor Progression.</a> Cell Rep. 18 (9), 2228-2242 (2017).</li></ol>

L'autore Ayano Kondo è un dipendente della Kyowa Hakko Kirin Co., Ltd.

Qui, abbiamo descritto un sistema di coltura semplice pH acido e il suo processo di valutazione. La combinazione dei tre metodi di acidificazione medio, vale a dire, concentrazione ridotta di bicarbonato, aggiunta di lattato e aggiunta di HCl, ci ha permesso di indagare a fondo il meccanismo di pH-risposta e di confrontare la risposta cellulare ad altro tumore condizioni microambientali come ipossia o nutriente inedia.
La chiave di questo metodo consiste nel determinare le concentrazi...

Il microambiente tumorale svolge un ruolo critico nella progressione del tumore e cancro cellula metabolismo1,2,3. Le cellule tumorali sono spesso esposti a condizioni quali ipossia, privazione nutriente e acida pH extracellulare (pHe). Tuttavia, il ruolo di pHe in progressione del tumore non è stato chiarito come ampiamente come ipossia o nutriente inedia. Il pHe nel tessuto del tumore può diventare acido, raggiungendo circa pH 6,84,5. Il pH acido nasce dall'aerobica ed anaerobica glicolitica escrezione di protoni (H+) e lattato di proliferazione del cancro cellule5,6.
Studi recenti hanno rivelato che acida indotta da pHe istone deacetilazione, ossidazione degli acidi grassi ed esocitosi dei lisosomi acidi per adattamento al severo ambiente acido7,8,9,10. Tuttavia, i meccanismi attraverso i quali l'acidificazione extracellulare influenza cancro comportamento e l'identità della chiave regolatori nel microambiente tumorale pH acido completamente non sono stati determinati. Inoltre, parecchi rapporti descritti vari media di pH acido, utilizzando poco chiare concentrazioni di bicarbonato, Tris, tubi e HEPES buffer o lattato e HCl, ma ci sono pochi rapporti per dimostrare la stabilità del rettificata media né completa confronto di diversi distinti acide cultura media7,8,9,10
Per delucidare i regolatori chiave ed i cambiamenti metabolici in cellule tumorali nel contesto dell'acidificazione extracellulare, abbiamo stabilito un modello semplice in vitro della coltura per mantenere un pHe acide ed ha esaminato il ruolo di pHe in cancro cellule11. Utilizzando questo metodo, abbiamo mantenuto un terreno di coltura acida con un pHe di 6.8 a 37 ° C inferiore al 5% CO2, usando le concentrazioni ridotte di bicarbonato nel terreno di coltura. pH 7.4 è stato utilizzato come normale e medio di controllo. Il pH medio era sostenuto per almeno 24 h e gradualmente diminuito di 72 h durante la coltura di cellule di cancro del pancreas PANC-1 e AsPC-1. Perché avanzata glicolisi accelera l'escrezione di non solo protoni, ma anche del lattato7,8,9, abbiamo anche stabilito un metodo di coltura che imita acidosi indotta da lattato di aggiunta del lattato, invece di ridurre il concentrazione di bicarbonato. Inoltre, indotta da HCl acido pHe nel medio permette di escludere la possibilità che risposte cellulari a pH acido di coltura non sono dovuti a una ridotta concentrazione di bicarbonato. Inoltre, utilizzando vari mezzi di comunicazione con un pH di 6.4 a 7.4 con concentrazione di bicarbonato differenti, possiamo valutare l'entità degli effetti di pHe su risposte cellulari.

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium&#8220;Nissui&#8221; 2</td>
			<td>Nissui</td>
			<td>05919</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher&#160;</td>
			<td>10438</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Wako</td>
			<td>191-01305</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine solution</td>
			<td>Thermo Fisher&#160;</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Mixed Solution(Stabilized)</td>
			<td>Nacalai</td>
			<td>09367-34</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5g/l-Tripsin/0.53mmol/l-EDTA Solution, with Phenol Red</td>
			<td>Nacalai</td>
			<td>32778-05</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5%-Trypan Blue Stain Solution</td>
			<td>Nacalai</td>
			<td>29853-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactic acid solution</td>
			<td>Sigma-Aldrich</td>
			<td>252476</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid solution</td>
			<td>Sigma-Aldrich</td>
			<td>H9892</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Plus Mini Kit</td>
			<td>QIAGEN</td>
			<td>74134</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript IV First-Strand Synthesis System</td>
			<td>Thermo Fisher&#160;</td>
			<td>18091</td>
			<td></td>
		</tr>
		<tr>
			<td>Power SYBR Green PCR Master Mix&#160;</td>
			<td>Applied Biosystems&#160;</td>
			<td>4368702&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PANC-1</td>
			<td>ATCC</td>
			<td>CRL-1469</td>
			<td></td>
		</tr>
		<tr>
			<td>AsPC-1</td>
			<td>ATCC</td>
			<td>CRL-1682</td>
			<td></td>
		</tr>
		<tr>
			<td>KMS-6</td>
			<td>JCRB Cell Bank</td>
			<td>JCRB0432</td>
			<td></td>
		</tr>
		<tr>
			<td>TIG</td>
			<td>JCRB Cell Bank</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MRC9</td>
			<td>ATCC</td>
			<td>CCL-212</td>
			<td></td>
		</tr>
		<tr>
			<td>HUVEC</td>
			<td>ATCC</td>
			<td>CRL-1730</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One Microvolume UV-Vis Spectrophotometer with Wi-Fi</td>
			<td>Thermo Fisher</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantStudio 5 Real-Time PCR System</td>
			<td>Thermo Fisher</td>
			<td>CRL-1682</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of an extracellular acidic ph culture system

Condizioni del microambiente tumorale, quali ipossia o nutriente inedia, giocano un ruolo critico nella progressione del cancro e malignità. Tuttavia, il ruolo di pH acido extracellulare in aggressività del tumore e del suo meccanismo sottostante non è stato ampiamente studiato rispetto alle condizioni ipossiche o nutriente inedia. Inoltre, un metodo di cultura ben definita per imitare il microambiente tumorale extracellulare acido completamente non è stato segnalato.
Qui presentiamo un metodo di coltura semplice in vitro per mantenere acida pH extracellulare utilizzando ridotto bicarbonato e aumento del lattato o HCl concentrazioni nel terreno di coltura. Il pH medio era sostenuto per almeno 24 h e gradualmente diminuito da 72 h dopo coltura di cellule di cancro del pancreas PANC-1 e AsPC-1. Tre condizioni distinte media acide in questo studio altamente sovraregolati geni pH-sensible a reagire come MSMO1, INSIG1e IDI1 rispetto ad ipossia o nutriente inedia. Il upregulation di questi geni può essere utilizzato come un indicatore di pH acido. Queste semplici tecniche sono benefiche per delucidare i meccanismi di fondo di malignità del tumore sotto microambiente tumorale acide. Di conseguenza, il nostro sistema di cultura acida pH extracellulare consente l'individuazione di risposte cellulari pH acido non solo nelle cellule tumorali, ma anche nelle cellule primarie, come le cellule tubolari renali, nei confronti di altri acidi disordini compreso, chetoacidosi diabetica, acidosi lattica, acidosi tubulare renale e acidosi respiratoria.

Per determinare la concentrazione di bicarbonato appropriato, abbiamo preparato DMEM al gamma di 0 - 8 mM NaHCO3 (concentrazione finale nel terreno di coltura) e riuscì a preparare terreni di coltura con pH che vanno da 6.4-7,4 (Figura 1). Abbiamo preparato DMEM con 8mm NaHCO3 (pH 7.4) come un mezzo di controllo e 2 mM NaHCO3 (pH 6,8) come un mezzo con pH acido secondo un precedente rapporto, affermando che il pH extracellula...

Watch this Scientific Journal Video about Establishment of an Extracellular Acidic pH Culture System at JoVE.com

Establishment of an Extracellular Acidic pH Culture System

Il microambiente tumorale acide gioca ruoli critici nella progressione del tumore. Per valutare gli effetti del pH extracellulare acido sul cancro cellule in vitro, abbiamo istituito sistemi di semplice cultura acida.

Istituzione di un pH extracellulare acido sistema cultura

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

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Unit 1

Fluid, Electrolyte, and Acid-Base Balance

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Video: Establishment of an Extracellular Acidic pH Culture System

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism 2, 3. Auxin activates a H+ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins 4, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins 5. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.
 To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 &deg;C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.

Cella di misura Estensione impianto a parete (Creep), indotta dal pH acido e da Alpha-espansina

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

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Biologia

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Un Ex-ovo Sistema di embrioni di pollo Cultura Adatto per applicazioni di imaging e Microchirurgia

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

Il metodo di coltura degli embrioni roditori intero utilizzando il Rotator tipo di sistema cultura Bottiglia

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

Un<em&gt; Ex vivo</em&gt; Cultura sistema per studiare lo sviluppo della tiroide

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

Sviluppo di un sistema di inserimento co-coltura di due tipi cellulari in assenza del cellula-cellula di contatto

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Ottica pH quantificazione di intracellulare in Drosophila melanogaster anatomista Epithelia con pH indicatore fluorescente geneticamente codificato

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Establishment of a Clonal Culture of Unicellular Conjugating Algae

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. Thus, it is extremely important to develop techniques to establish clonal cultures. In this article, we demonstrate the establishment of clonal cultures of a conjugating alga. Water samples are collected from the field. Subsequently, cells are isolated using a glass capillary pipette, placed in media, and grown under conditions suitable for generating a clonal culture.

In this article, we demonstrate the establishment of clonal cultures of unicellular conjugating algal species collected from a natural field site.

Creazione di una cultura clonale di alghe unicellulari coniugazione

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

Misurazione della confluenza di iPSC utilizzando un sistema di imaging automatizzato.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.

Coltura di organoidi intestinali 3D di maialino da cripte epiteliali crioconservate e creazione di monostrati cellulari

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

Isolamento avanzato delle vescicole extracellulari batteriche per la multi-omica del microbiota

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.
This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.
Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

Autore in primo piano: Accelerare la ricerca sulla separazione e l'eterogeneità delle vescicole extracellulari batteriche 

This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.