Biz Division genom bilim ve laboratuvar üyeleri teşekkür sistemleri Biyoloji ve tıp, RCAST, Tokyo Üniversitesi için. Biz özellikle teşekkür Dr H. Aburatani, Dr. T. Kodama, (LSBM, RCAST, Tokyo Üniversitesi), Dr. K. Tomizuka, Dr. T. Yoshida ve Dr. A. Kunisato (Kyowa Hakko Kirin Co, Ltd) yararlı tartışmalar ve destek. Bu eser kısmen için genç bilim adamı (A) (26710005, T.O.), yenilikçi alanları (26116711 ve 16 H 01567, T.O.) bilimsel araştırma için Grant-in-Aid ve Grant-in-Aid için zorlu Grant-in-Aid tarafından desteklenmiştir Araştırmacı araştırma (16K 14605, T.O.) Milli Eğitim Bakanlığı, kültür, spor, bilim ve teknoloji Japonya, Takeda Bilim Vakfı (to), kanser araştırmaları (to) Kobayashi temeli ve kanser araştırmaları için proje ve Terapötik evrim (P-oluşturma) ve yenilikçi kanserle savaş Japonya ajansı tıbbi araştırma ve geliştirme, AMED (to) için pratik araştırma.

1. asidik kültür orta hazırlanması
<ol><li>
Denetim (pH 7,4) hazırlanması ve düşük-pH (pH 6.8) kültür orta
	<ol>
<li>4.75 g L-glutamin olmadan DMEM tozu 500 mL su içinde çözülür.</li>
		<li>0,33 M NaHCO3 çözüm basınç sıkı bir şişe hazırlayın. Dikkat: NaHCO3 ısıtmalı ve termal olarak çürümüş CO2 gaz yayar bu yana bir basınç sıkı şişe kullanmak önemlidir.</li>
		<li>Otoklav orta ve çözüm. Bu arabellekleri 25 ° C'ye soğutmak izin</li>
		<li>200 mM L-glutamin, penisilin-streptomisin, 5 mL 5 mL ekleyin ve 50 mL fetal sığır serum DMEM 500 ml bikarbonat olmadan.</li>
		<li>Denetim (pH 7,4) orta için 0,33 M NaHCO3 çözüm DMEM 100 mL bikarbonat olmadan başına 2,4 mL ekleyin. Düşük-pH (pH 6.8) orta için 0,33 M NaHCO3 çözüm DMEM 100 mL bikarbonat olmadan başına 0.6 mL ekleyin.</li>
		<li>Altında %5, kuluçka 37 ° C'de 24 saat sonra orta pH kontrol CO2. Not: NaHCO3 çözüm miktarı kuluçka ve hava basıncı CO2 içeriğe bağlı olarak farklı olabilir orta pH bağlı olarak ayarlanması gerekir. İdeal olarak, ilk kez, NaHCO3 çözüm için DMEM eklemek için uygun miktarını belirlemek için çeşitli bikarbonat konsantrasyonu ile orta pH ölçmek daha iyi.<ol>
<li>Hemen CO2 kuluçka kültür çanak çıkardıktan sonra kültür orta toplamak ve pH metre kullanarak pH ölçmek. Orta sıcaklık bir su banyosu kullanarak 37 ° C'de tutun. Orta pH bağımsız olarak üç kez ölçmek. Not: NaHCO3 çözüm 4 ° C'de muhafaza edilmelidir ve her ortamın taze hazırlanmalıdır. Bu çalışmada kullanılan piyasada bulunan ürünler Malzemeler tabloiçinde listelenir.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Laktat kaynaklı veya HCl kaynaklı asidik kültür ortamı hazırlama
	<ol>
<li>74 µL laktat çözüm veya 1 M HCL 125 µL denetim orta (1.1.5 adımda hazırlanan) 100 mL ekleyin.</li>
		<li>Altında %5, kuluçka 37 ° C'de 24 saat sonra orta pH kontrol CO2. Not: Laktat veya HCl bir çözüm de kuluçka ve hava basınç CO2 içeriğe bağlı olarak farklı olabilir orta pH göre ayarlanması gerekir. İdeal olarak, seri laktat veya HCl konsantrasyonu için ortamın pH istenen ayarlamak için uygun miktarını belirlemek ile orta pH ölçmek iyidir.</li>
	</ol>
</li>
</ol>2. hasat ve hücreleri tedavi
<ol><li>Altında %5 CO2 gerçekleştirmeden önce en az 1 hafta deneyler 37 ° C'de hücreler yetiştirilmesi başlatın. Not: Bu çalışmada kullanılan hücre hatları Malzemeler tabloiçinde listelenir.</li>
	<li>% 70-90 confluency ulaştığınızda geçiş hücreleri. Hücreleri Konfluent olmak izin vermez. Orta 2-3 günde günlük ekimi sırasında değiştirin.</li>
	<li>Hücreleri 5 mL PBS ekleyerek yıkayın. PBS Aspire edin ve 1 mL tripsin gibi ayırmayı enzim ekleyin.</li>
	<li>Hücreleri yuvarlanır ve ayırmak başlamak kadar 37 ° C'de kuluçkaya.</li>
	<li>5 mL kültür orta de müstakil hücrelere ekleme ve enzim devre dışı bırakın. 15 mL tüp ve 300 x g. aspiratı süpernatant adlı 3 dk santrifüj hücre süspansiyon aktarmak ve kültür orta hücrelerle yeniden askıya alma.</li>
	<li>Bir hemasitometre ve Trypan-mavi kullanarak canlı hücreleri saymak.</li>
	<li>Tohum 5.0 x 105 hücreleri/iyi bir 10 cm tabak orta 10 ml. 37 ° c altında %5 24 h için hücreleri kuluçkaya CO2.</li>
	<li>Kültür orta Aspire edin, 5 mL PBS hücrelerle yıkama ve 10 mL asidik kültür orta bölüm 1'de hazırlanan ekleyin. 37 ° c altında %5 24 h için hücreleri kuluçkaya CO2.</li>
</ol>3. RNA izolasyon
<ol><li>Kültür orta Aspire edin ve hücreleri 5 mL PBS ile yıkayın. Hücrelerin RNA ayıklama reaktif ekleyerek bozabilir ( Malzemeleri tablolarıgörmek).</li>
	<li>Çanak kısaca kazımak, RNA ayıklama reaktif bir pipet ile kaldırın ve RNA ayıklama reaktif/hücre 1,5 mL tüp lysate mevduat. 5 min için oda sıcaklığında bırakın.</li>
	<li>250 µL kloroform ekleyin ve 5 min için oda sıcaklığında yaklaşık 15 s. bırak ve ≥8, 000 x g 5 min için santrifüj tüpü şiddetle çalkalanır.</li>
	<li>Bir pipet kullanarak sulu faz dikkatli bir şekilde çıkarın. Sulu faz bırak. Başka bir 1,5 mL tüp içinde yerleştirin.</li>
	<li>Sulu faz 550 µL isopropanol ekleyin ve karışımı yavaşça. 5 dakika santrifüj (≥20, 000 x g) maksimum hızda süreyle oda sıcaklığında 4 ° C'de 20 dk için bırakın</li>
	<li>İsopropanol dökün ve 1 mL % 75 etanol DEPC su tedavi ekleyin. Hafifçe karıştırın. 8000 x g 5 min için de santrifüj kapasitesi.</li>
	<li>Etanol ve granül kurumasına izin pipet.</li>
	<li>DEPC yaklaşık µL 15-25 (verim) bağlı olarak su RNA Pelet tedavi ekleyin. İyice karıştırın.</li>
	<li>260 OD ölçerek izole RNA konsantrasyonu ölçmek bir Spektrofotometre kullanarak nm.</li>
</ol>4. cDNA sentezi
<ol><li>PCR reaksiyon tüp içinde aşağıdaki parçaları birleştirmek: 5 µg toplam RNA, 1 µL Oligo dT (50 µM) veya astar karışımlarda (60 µM), 1 µL 10 mM dNTP ve 14 µL toplam hacmiyle nükleaz ücretsiz su.</li>
	<li>Mix ve kısaca bir küçük benchtop santrifüj kullanarak bileşenlerini santrifüj kapasitesi (~ 5 s). 65 ° C'de 5 min için örnek RNA/astar denatüre Kısaca spin (~ 5 s) küçük bir benchtop kullanarak santrifüj kapasitesi ve örnek derhal en az 1 dk. için buz koymak.</li>
	<li>Aşağıdaki bileşenleri ekleyin: 4 µL 5 ters transkripsiyon arabellek x, 1 µL transkriptaz (200 U/µL), 1 µL RNase inhibitörü (40 U/µL). Not: Piyasada kitleri örnekleri Malzemeler tablo gösterilir
</li>
	<li>Tüp, kap mix ve kısaca belirtilen içeriğini bir küçük benchtop santrifüj santrifüj kapasitesi (~ 5 s). 10 dk 50-55 ° C kombine tepki karisimin kuluçkaya.</li>
	<li>Reaksiyon 10 dk 80 ° C'de kuluçka tarafından devre dışı bırakabilirsiniz.</li>
</ol>5. gerçek zamanlı PCR kullanarak asit duyarlı gen ekspresyonu ölçümü
<ol><li>Aşağıdaki bileşenler, 384-iyi tepki plaka birleştirmek: 1 µL şablon cDNA, 0,75 µL 10 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 X 0.3 µL 50 µM ileri astar, 0.3 µL 50 µM ters astar, 1 µL Taq polimeraz, 2.5 µL 10 x Taq polimeraz arabellek, 2.5 µL 20 mM dNTP, 16.65 µL su. Not: Piyasada kitleri örneği Malzemeler tablogösterilmiştir. Astar listesi Tablo 1' de gösterilen. 96-iyi tepki plaka da bu adımda kullanılabilir. Diğer probları (örneğin, Taqman sonda) de kullanılabilir.</li>
	<li>Deneme ve gerçek zamanlı PCR sistemi aşağıdaki PCR programa ayarlayın: 50 ° C için 2 dk, 1 döngüsü; 10 dk, 1 döngüsü için 95 ° C; 95 ° C 15 s ve 60 ° C için 1 dakika, 40 döngüleri; 95 ° C 15 s, 1 döngüsü; 60 ° C 15 s, 1 döngüsü; 95 ° C 15 s, 1 döngüsü.</li>
	<li>Yer plaka qPCR alet. Programı başlatın. Not: pH duyarlı genlerin MSMO1Upregulation, INSIG1ve IDI1 tarafından protein düzeyleri ölçülebilir.</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>

Yazar Ayano Kondo bir çalışandır, Kyowa Hakko Kirin Co., Ltd.

Burada, bir basit asidik pH kültür sistemi ve değerlendirme sürecinin açıklanan. Orta asitleştirme, Yani, düşük bikarbonat konsantrasyonu, laktat ek ve HCl ek olarak, üç yöntemlerin bileşimini pH-yanıtlama mekanizması iyice araştırmak ve hücresel yanıt diğer tümör olarak karşılaştırmak için bize etkin hipoksi veya besin açlık gibi microenvironmental koşulları.
Bu yöntem, kültür orta uygun konsantrasyonlarda bikarbonat, laktat ve HCl belirlemek için ...

Tümör microenvironment tümör ilerleme ve kanser hücre metabolizması1,2,3kritik rol oynar. Kanser hücreleri genellikle hipoksi, besin yoksunluğu ve asidik ekstrasellüler pH (pHe) gibi koşullara maruz kalır. Ancak, tümör ilerleme pHe rolü gibi kapsamlı açıklık olmuştur değil hipoksi veya besin açlık olduğu gibi. Tümör dokusunda pHe yaklaşık pH 6.84,5ulaşan asidik hale gelebilir. Asidik pH aerobik ve anaerobik glycolytic atılımı proton (H+) doğar ve laktat Proliferasyona kanser tarafından5,6hücreler.
Son yıllarda yapılan çalışmalarda ortaya o asidik pHe kaynaklı Histon deasetilasyonu, yağ asidi oksidasyon ve ağır asidik ortamı7,8,9,10adaptasyon için asidik organellerin ekzositozu. Ancak, mekanizmalar aracılığıyla hangi hücre dışı asitleştirme kanser davranış ve düzenleyiciler asidik pH tümör microenvironment içinde tam olarak tespit anahtar kimliğini etkiler. Ayrıca, çeşitli raporlar bikarbonat, Tris, borular ve HEPES tampon veya laktat ve HCl belirsiz konsantrasyonları kullanarak çeşitli asidik pH medya açıklanan, ancak kararlılığını ayarlanan orta ne de kapsamlı göstermek için birkaç rapor vardır: birkaç farklı asidik kültür media7,8,9,10 karşılaştırılması
Anahtar düzenleyiciler ve kanser hücrelerinin hücre dışı asitleştirme bağlamında metabolik değişiklikler aydınlatmak için kurulan bir asidik pHe korumak için basit vitro kültür manken ve pHe kanser hücreleri11' deki rolü inceledi. Bu yöntemi kullanarak, biz tutulan 37 ° c altında %5 6,8 pHe ile asidik bir kültür orta düşük bikarbonat konsantrasyonu kültür ortamı kullanarak CO2. pH 7.4 normal olarak kullanılan ve orta kontrol. Orta pH en az 24 saat sürekli ve 72 h PANC-1 ve AsPC-1 pankreas kanseri hücre kültür sırasında yavaş yavaş azaldı. Gelişmiş Glikoliz sadece da laktat7,8,9proton atılımı hızlandırır çünkü aynı zamanda asidoz laktat kaynaklı yerine laktat ekleme azaltarak tarafından taklit eden bir kültür yöntemi kurdu bikarbonat konsantrasyonu. Ayrıca, HCl kaynaklı asidik pHe orta asidik pH kültür ortamına hücresel yanıt bikarbonat azaltılmış bir konsantrasyon nedeniyle değildir olasılığını dışlamak için bize izin verir. Ayrıca, 6.4-7,4 farklı bikarbonat konsantrasyonu ile pH ile çeşitli medya kullanarak, biz ölçüde hücresel yanıt pHe efektleri değerlendirebilir.

<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

Hipoksi veya besin açlık gibi tümör microenvironment şartları kanser ilerlemesi ve malignite kritik rol oynarlar. Ancak, asidik ekstrasellüler pH tümör saldırganlık ve onun altında yatan mekanizma rol kapsamlı hipoksik veya besin açlık koşulları ile karşılaştırıldığında araştırılmamıştır. Buna ek olarak, asidik ekstraselüler tümör microenvironment taklit etmek için iyi tanımlanmış kültür yöntemi tam olarak bildirilmemiştir.
Burada asidik ekstrasellüler pH kullanarak bikarbonat ve artan laktat veya HCl konsantrasyonu kültür orta azaltılmış korumak için basit vitro kültür yöntemi mevcut. Orta pH en az 24 saat sürekli ve yavaş yavaş 72 kültür PANC-1 ve AsPC-1 pankreas kanseri hücrelerinin takip h tarafından azalmıştır. Bu çalışmada üç ayrı asidik ortam koşullarında son derece hipoksi ya da besin açlık. MSMO1, INSIG1ve IDI1 gibi upregulated pH-yanıt veren genler göre Bu genlerin upregulation asidik pH bir işareti olarak kullanılabilir. Bu basit teknikler asidik tümör microenvironment altında tümör malignite temel mekanizmaları aydınlatmak yararlıdır. Bu nedenle, hücre dışı asidik pH kültür sistemimiz hücresel asidik pH yanıt sadece kanser hücrelerinin zamanda kadar asidik diğer bozukluklar da dahil olmak üzere, Diyabetik ketoasidoz ile ilgili olarak renal tübüler hücreler gibi Primer hücre keşfi sağlar, Laktik asidoz, renal tübüler asidoz ve solunum asidozu.

Uygun bikarbonat konsantrasyonu belirlemek için DMEM aralığı 0 - 8 mM NaHCO3 (kültür orta son konsantrasyonu) hazırlanmış ve 6.4-7.4 (şekil 1) arasında değişen pH ile Kültür medya hazırlanmasında başarılı oldu. DMEM ile 8 mM NaHCO3 (pH 7,4) olarak bir denetim orta ve asidik pH ile 2 mM NaHCO3 (pH 6.8) bir ortam olarak ekstrasellüler pH pH 6.8 solid tümör4,<sup class="xr...

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

Asidik tümör microenvironment tümör ilerlemesinde önemli rol oynar. Kanser hücreleri in vitro olarak asidik ekstrasellüler pH etkilerini değerlendirmek için basit asidik kültür sistemleri kuruldu.

Bir hücre dışı asidik pH kültür sistemi kurulması

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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14.5K Görüntüleme.  Kyowa Hakko Kirin Co., Ltd. Asidik tümör microenvironment tümör ilerlemesinde önemli rol oynar. Kanser hücreleri in vitro olarak asidik ekstrasellüler pH etkilerini değerlendirmek için basit asidik kültür sistemleri kuruldu.

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.

Asidik pH ve Alpha-Expansin İndüklenmiş Ölçüm Bitki Hücre Duvarı Uzantısı (Creep)

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 ...

Biyoloji

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.

Bir Eski-ovo</emGörüntüleme ve Mikrocerrahi Uygulamaları için uygundur> Tavuk Embriyo Kültürü Sistemi

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.

Rotator-tipi Şişe Kültür Sistemi kullanılarak Kemirgen Tüm Embriyo Kültürü Yöntemi

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.

Bir<em&gt; Ex vivo</em&gt; Kültür Sistemi Tiroid Geliştirme Çalışması

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.

Hücre-Hücre İletişim Yokluğunda iki Hücresel Türleri Ekleme Co-kültür Sisteminin Geliştirilmesi

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.

Drosophila melanogaster Malpighi tübül epiteli optik Quantification intrasellüler pH floresan genetik olarak kodlanmış pH göstergesi

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.

Tek hücreli alg Conjugating klonal bir kültür kurulması

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.

Otomatik Görüntüleme Sistemi kullanarak iPSC'lerin Birleşme Noktasının Ölçülmesi.

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.

Kriyokorunmuş Epitel Kriptlerinden Domuz Yavrusu Bağırsak 3D Organoidlerinin Kültürü ve Hücre Tek Katmanlarının Oluşturulması

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

Mikrobiyota Multi-Omikler için Gelişmiş Bakteriyel Hücre Dışı Vezikül İzolasyonu

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.

Yazar Spotlight: Bakteriyel Hücre Dışı Veziküllerin Ayrılması ve Heterojenliği Üzerine Araştırmaların Hızlandırılması 

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.

Methods Article

Bir hücre dışı asidik pH kültür sistemi kurulması