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1. çeşitli memeli hücre satırlarını anaerobik kültür için ortam hazırlamak
<ol><li>
Anoksik hücre kültür25tam PS-74656 orta yapmak.
	<ol>
<li>17.25 g nitrit 50 ml distile deiyonize su ile çözülerek steril nitrit hisse senedi çözüm (5 M; 100 x) yaptıktan sonra bu sterilize etmek için filtre.</li>
		<li>Nitrit hisse senedi çözüm düşük glikoz DMEM (glikoz 1 g/L) 50 mL başına 0.5 mL 110 mg/L L-glutamine, 584 mg/L sodyum pyruvate ve % 10 fetal Sığır serum ile ekleyin (FBS; ısı-inaktive olur). Not: FBS konsantrasyonu % 10 toksik büyük kullanımı. Not, bir kanser hücre satır test nitrit (MDA-MB-231, meme kanseri hücre satır) ile media için düşük tolerans vardı ve böylece, nitrit, yokluğunda büyüdü. Buna ek olarak, PS-74656 orta test edildi tüm hücre satırlarını büyüme desteklenen rağmen (n = 9), belirli hücre hatları (Örneğin, Vero hücreleri) sergilenen daha yüksek hücre konsantrasyonları ve yüksek glikoz (4,5 g/L) DMEM ile daha yüksek bir canlılık içinde PS-74656 25.</li>
		<li>Orta 20 mL steril 50 mL konik santrifüj tüpü yerleştirin. Kap sıkın değil; gevşek olmalıdır.</li>
		<li>Orta de-gaz tüp bir vakum çan kavanoza koyarak bir yaklaşık 45 ° açılı orta yüzey alanı en üst düzeye çıkarmak için.</li>
		<li>Bir vakum pompası veya eşdeğeri, oda sıcaklığında kullanarak bir vakum uygulanır. Kabarcıklar artık (24-48 h) gözlenir kadar vakum korumak.</li>
		<li>Kavanozdan tüpü çıkarması ve mühürleme hava giriş Orta içine en aza indirmek için tüp kap, hemen kapatın.</li>
		<li>Tüp ticari anaerobik odasına koyun.</li>
		<li>Tüp kapağı gevşetin ve atmosfer bu en az 24 saat için odasında anaerobik gaz karışımı ile equilibrate için tüp izin vermek. Not: Bu kullanılana kadar orta odasında kalır. Tüp işleminizi, floş hızlandırmak için steril transfer pipet kullanarak anoksik gaz karışımı ile en az 3 x H2, CO2ve N oluşan anoksik gazı ile doldurulmuş2.
</li>
		<li>Tüm borular (10-15 dk) tüp mühürleme ve odasında yerleştirmeden önce yukarıda açıklandığı gibi anaerobik odasında yerleştirilen degas. Kullanmadan, 1.5-2 mL transfer pipetler kullanarak anaerobik gaz karışımı ile temizleme veya en az 3 x ile anoksik temizlenen pipet İpuçları gaz.</li>
		<li>Odasında anaerobik iken, steril degassed mikro-santrifüj tüpü için orta 100 µL kaldırmak ve bir oksijen sondası odası içinde kullanarak çözünmüş oksijen için test edin. Not: Oksijen elektrot kullanmadan üreticinin Protokolü önce kalibre edilmiş. Düzgün bir şekilde degassed ve denge anaerobik gaz karışımı medya okumaları 0. Dikkat: 0.3 ppm oksijen okumaları, orta equilibrate için daha fazla zaman gerekli olduğundan anaerobik odası medyada kuluçkaya devam.</li>
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</ol>2. anoksik çeşitli memeli hücre satırlarını tarımı
<ol><li>
Belirlenen gün -1
	<ol>
<li>Hücreler (37 ° C, % 5 CO2) % 90 izdiham hücreleri yeterli sayıda üç 24-şey tabak (% 80 izdiham) sağlamak anoksik ve normoxic kültür kontrolü için bir T150 şişeye büyümeye. Not: Bu çalışma için HeLa 229 ve Vero hücreleri kullanılmıştır. Bu iletişim kuralı ayrıca test ve büyüme ve yedi diğer hücre hatları25bakımı için başarılı oldu.</li>
		<li>Orta balonun aspirasyon tarafından kaldırmak ve HBSS 10 mL ile yıkayın.</li>
		<li>Orta tripsin 5 mL ile değiştirin. Hücreleri görsel olarak plastik ayırmak kadar şişeye kışkırtmak. Tripsin çoğunu Aspire edin sonra şişeye yapışık hücreleri çıkarmak için dokunun. Yüksek glikoz DMEM 10-15 mL ekleyin (4,5 g/M glikoz, 110 mg/L L-glutamine, % 10 FBS, gentamisin 50 µg/mL) tripsin devre dışı bırakabilirsiniz için.</li>
		<li>Tüm hücre kümeleri bozulduğu kadar 25 mL pipet kullanarak hücreleri triturate.</li>
		<li>Hücreleri saymak ve standart hemasitometre ve trypan mavi boya hariç tutma yöntemini veya otomatik eşdeğer kullanarak canlılığı belirlemek.</li>
		<li>Yüksek glikoz DMEM hücrelerde askıya alma 105 hücre/mL x 2,24 hücre yoğunluğu için yukarıda belirtildiği gibi.</li>
		<li>24-iyi doku kültürü plaka wells için hücre süspansiyon 1 mL ekleyin (2,24 x 105 hücreleri/iyi; % 80 izdiham). Anoksik kültür ve başka bir denetim normoxic kültür için tohum 1 plaka.</li>
		<li>Normoxic koşulları (%5 CO2) için 24 saat içinde 37 ° C'de hücreler kuluçkaya. Not: Hücreleri çeşitli iyi ölçekli levha kaplama. Bakım tamamen atmosferik gaz temizlemek için icra sürece ancak hücreleri şişeler anoksik büyüme için tohum oksijen getirilmesi riskini sisteme çalışır.</li>
	</ol>
</li>
	<li>
Belirlenen gün 0
	<ol>
<li>Normoxic kuluçka 24 saat sonra plakaları anoksik büyüme için anaerobik odasına (ticari anaerobik odası; yerleştirin. Şekil 1).</li>
		<li>Hemen hangi anaerobik odasına 24 h için iklime alıştırılacağı antibiyotik ücretsiz de-gazla PS-74656 orta orta değiştirin. Nemli bir havlu ile bir kapta plaka yerleştirin ve gevşek nem korumak için kapatın. Not: Tabakları normoxic büyüme için aynı şekilde tüm tedaviler atmosferik koşullar altında yapılır özel durum ele alınır.</li>
	</ol>
</li>
	<li>
Belirlenen gün 1
	<ol>
<li>Gün 1, anaerobik Kuluçka kuluçka %10 H2, % 10 CO2ve % 80'i N2standart anaerobik gaz karışımı içeren ticari anaerobik odasında 24 saat sonra başlar.</li>
		<li>Hücreleri çeşitli süreler için kuluçkaya.</li>
		<li>Zaman içindeki renk değişimi için anaerobik orta izlemek. Not: Kırmızı-turuncu pembe bir renk kayması zaman orta değiştirmek için sinyal gönderir. Bu gerçekleşmesi için 2 hafta sürebilir. Bir renk vardiya orta kırmızı-turuncu sarı bir bakteriyel kontaminasyon varlığını gösterir.</li>
		<li>Orta için pembe kırmızıdan değiştiğinde, runagate hücreleri tarafından aspirasyon kaldırın.</li>
		<li>Yer degassed ve anaerobik gaz runagate hücrelerde emişli harcanan ortamıyla microfuge tüpler equilibrated, tüpler mühür güvenli ve sonra onları (326 gx; 10 dakika oda sıcaklığında) santrifüj kapasitesi emin yaparken yakın. Hemen iyi emişli ortamı yeni degassed PS-74656 orta 1 mL ile değiştirin.</li>
		<li>Microfuge tüp pelleted hücrelerle açmadan önce anaerobik odası yerleştirin. Harcanan orta Aspire edin ve taze degassed PS-74656 orta ve 1 ml microfuge tüp toplam hacmi ile değiştirin.</li>
		<li>Hücreleri tüm tüpünden 24-iyi doku kültürü plaka bir kuyuda dönün.</li>
	</ol>
</li>
</ol>3. fenotipik hücre farklılaşması anaerobik kültürlü hücrelerinin mikroskopisi tarafından değerlendirilmesi
<ol><li>
Anaerobik odası ise, plaka anaerobik gaz karışımı ile boşaltılan resealable kutusuna yerleştirin ve kutusunu kapatın. Not: Nem ve plaka kurumasını önlemek için nemli kağıt havlu kutusu içermelidir. Onlar ince olduğundan Mikroskopi için sandviç paketi en iyi iş.<ol>
<li>Hücreleri mikroskop altında gözlemlemek için tamamen çanta mühür ve odasından kaldırın.</li>
		<li>Dikkatle plastik torba plaka üzerinde streç, bir ters faz kontrast mikroskobu ile fotoğraf makinesi eki kullanarak hücreleri incelemek ve photomicrographs farklı büyütme oranlarında al.</li>
		<li>Plaka mühürlü çantada odasına dönün ve kuluçka 2.3.3. adımda anlatıldığı gibi devam etmemiz gerekiyor.</li>
		<li>Süspansiyon içinde olan runagate hücreler ayrı ayrı analiz için 2.3.5. adımda anlatılan adımları izleyin. Not: Bu hücreler daha sonra aerobik test etmek için kullanılabilir veya bu belirli nüfusu testleri için anaerobik odasına döndü.</li>
	</ol>
</li>
</ol>

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Yazarlar ifşa gerek yok.

Bu yöntem için uzun bir süre oksijen yokluğu kültürlü ilk memeli hücreleri temsil eder. Geçerli gözlem dayalı, anoksik büyüme özelliği üzerinden fermantatif olmayan bir yol nereye fenotip sapma anaerobik büyüme sonuçlandı memeli hücre hatları28arasında evrensel gibi görünüyor. Bu test tüm hücre hatlarında gözlendi. Anaerobik ekimi ile artan oranlarda hücreleri yuvarlak oldu, süspansiyon gibi nüfus geliştirilen ve çoğaltmak başardık. Orta hücre tipi i...

Solid tümör, kök hücre nişler ve bu mukozal yüzeylerde astar hücrelerden düşük oksijen düzeylerinde, kanda oksijen azlığı1,2,3,4de dahil olmak üzere ortamlarda adlı biri yok. Normal fizyolojik Birleşik Devletleri, hipoksi, kanda oksijen azlığı (oksijen olmaması)5,6ötesinde oksijenasyonu değişir. Atmosferik oksijen olumsuz memeli hücre çoğaltma etkiler ve o vitro hücre büyümesini tükenmiş oksijen koşullar altında optimize edilebilir gerçekleşme 1970'lerde bildirildi. Richter vd. 7 1-%3 oksijen düzeyleri (hipoksi) kaplama verimliliği ile karşılaştırıldığında atmosferik oksijen (% 20) gelişmiş gösterdi. İnsan diploit hücre ömrü hipoksik kültür koşulları8' de genişletilir.
Vivo, hipoksik koşullar ortaya oksijen depoları tükenmiş (Örneğin, yoğun egzersiz sırasında), olduğunda neyin ATP üretimi fermantasyon (anaerobik solunum) için gelen aerobik solunum kas iskelet ile açık olduğundan laktik asit9son ürünü. Patolojik olarak, kanserli tümörleri, iç içinde kitle için zavallı vaskülarizasyon nedeniyle anoksik hipoksik10tümördür. Tümör iç sınırlı perfüzyon etkisi bağımsız olarak zorunlu anaerobik1tarafından kolonize tümör iç tarafından doğrulanır. Mechanistically, tümör hücre hayatta hipoksi hipoksi-indüklenebilir faktörü 1-alfa Gen ifadesinin yalnızca bağımlı olduğu düşünülmektedir (HIF1-alfa), ilk spontan yanıt hipoksi4,11 olduğu , 12. HIF1-Alfa HIF1bağlamak Isı şok proteinleri tarafından hipoksik koşullarda indüklenen-Alfa organizatörü ve upregulate Gen transkripsiyonu12. Bu ısı şok proteinleri tümör hipoksik microenvironment görülen çeşitli fenotipleri indüksiyon yardım inanılıyor. Bu fenotipleri hücre zarı Glikoz taşıyıcı artan bir ifadesidir ve glikoliz (Warburg etkisi)13oranı sergi. Mitokondriyal Oksidatif fosforilasyon fermantasyon laktat anahtarından sonucudur.
Anoksik hayatta kalma glikoz hayatta kalma fenomen14,15desteklemek için alternatifler de kullanabilir. En iyi okudu memeli yaklaşık 20 dakika oksijen ile fruktoz tahrik glycolytic fermantasyon yolu14olmadan yaşayabilir kör fare örnektir. Alternatif bir uyum içinde belirli balık oluşur (Örneğin,'önemli ölçüde daha uzun hayatta kalabileceği sazan [Carassius sp.], zaman glikoliz etanol ile terminal yan ürün kullanarak dönemleri)15. Her iki durumda da, fermantasyon oksijen yokluğunda yaşama etkinleştirme metabolizma kullanıyor. Geçerli anoksik hayatta kalmak için bu kadar uzun HIF1hipotezdir-Alfa hipoksi, mitokondrial solunum sırasında aktif oksijen ihtiyacını ortaya çıkar olmadan anaerobik16altında. Ayrıca, hücreleri hücre hayatta kalma17' ye zararlı olarak kanıtlamak olabilir oksidatif stres önlemek bu yana fermantatif bir yol kullanımı hipoksik/anoksik hayatta kalmak için tümör hayatta kalma artırır öne ise. Bu postülatı cardiomyocytes, hipoksi tümör hücre17tarihinde yerleştirilen oksidatif stresi azaltır gösteren bir çalışmada tarafından desteklenmektedir.
Bugüne kadar bir fermantatif yol anoksik memeli hücre hayatta kalmak için temel doğası literatürde, kusura bakma ama, bir yetersizlik kültür memeli hücreleri oksijen 3 günden uzun olmaması için büyük ölçüde kökleşmiş. Ancak, bakteriler glikoliz anaerobik hayatta kalmak için bir alternatif oluşur. Bazı bakteriler, azot veya sülfat (arasında diğer bileşikleri) yokluğunda sitokrom oksidaz sistemi oksijen18elektron alıcısı olarak hizmet verebilir. Her ne kadar son evrensel ortak atadan uzaklaşan beri paralel olarak bakteriyel ve ökaryotik evrim oluştu, bu mitokondri 1.542 milyon yıl önce okyanuslarda19 oxygenation hücrelere enerji sunuyorlardı tahmin edilmektedir . Hücreler zaman oksijen yokluğu 3 gün daha uzun süreler için işlev olabilir varsaymak makul çünkü araştırmacılar izole mitokondri elektron alıcısı olarak nitrit ile oksijen yokluğu ATP üretebilir göstermiştir 20 , 21 , 22 , 23. Bu çalışmada açıklanan metodoloji anaerobik memeli hücre büyüme çok sayıda hücre hatları için bir yardımcı program vardır.

<table border="0" cellpadding="0" cellspacing="0" style="width:709px;" width="709">
	<tbody>
		<tr height="147">
			<td height="147" style="height:147px;width:255px;">Whitney A35 anaerobic chamber</td>
			<td style="width:149px;">Don Whitley Scientific&#160;</td>
			<td style="width:139px;">Microbiology International</td>
			<td style="width:167px;">The ability to remove the front of the chamber makes it easy to put instruments inside without concern about getting them through the portals.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">CO2 incubator</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:139px;">3531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tissue Culture Hood</td>
			<td style="width:149px;">Labconco</td>
			<td style="width:139px;">DO55735</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">pipet aid</td>
			<td style="width:149px;">Drummond</td>
			<td style="width:139px;">4-000-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">sterile transfer pipets</td>
			<td style="width:149px;">Santa Cruz</td>
			<td style="width:139px;">sc-358867</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">50cc sterile conical centrifuge tubes</td>
			<td style="width:149px;">DOT</td>
			<td style="width:139px;">451-PG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">vaccum jar</td>
			<td style="width:149px;">Nalgene 111410862889</td>
			<td style="width:139px;">BTA-Mall 5311-0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DMEM high glucose (4.5 g/L)</td>
			<td style="width:149px;">CellGro</td>
			<td style="width:139px;">10-017-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DMEM low glucose (1 g/L)</td>
			<td style="width:149px;">CellGro</td>
			<td style="width:139px;">10-014-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">FBS</td>
			<td style="width:149px;">VWR</td>
			<td style="width:139px;">1500-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">HBSS</td>
			<td style="width:149px;">VWR</td>
			<td style="width:139px;">20-021-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">trypsin</td>
			<td style="width:149px;">VWR</td>
			<td style="width:139px;">25-052-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">gentamicin</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:139px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">sterile pipets</td>
			<td style="width:149px;">CellTreat</td>
			<td style="width:139px;">229210B, 229205B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tissue culture flask (T75 or T150)</td>
			<td style="width:149px;">Santa Cruz</td>
			<td>sc-200263, sc-200264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">24 well tissue culture treated dishes</td>
			<td style="width:149px;">DOT</td>
			<td style="width:139px;">667124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">glad ziplock sandwich bags</td>
			<td style="width:149px;">Ziploc</td>
			<td style="width:139px;">Costco</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">inverted phase microscope (10, 20 40x objectives with camera mont)</td>
			<td style="width:149px;">Nikon Eclipse</td>
			<td style="width:139px;">TS100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">trypan blue</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:139px;">T10282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">hemocytometer</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:139px;">C10283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Countess Automated Cell Counter</td>
			<td style="width:149px;">Life Technologies</td>
			<td style="width:139px;">AMQAF1000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Rainin microtiter pipets</td>
			<td style="width:149px;">Mettler Toledo</td>
			<td style="width:139px;">Rainin Classic Pipette PR-100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">microtiter tips</td>
			<td style="width:149px;">Santa Cruz Biotechnology</td>
			<td style="width:139px;">sc-213233 &#38; sc-201717&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">test tube rack (50cc tubes)</td>
			<td style="width:149px;">The Lab Depot</td>
			<td>HS29050A</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:255px;">sodium nitrite</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:139px;">https://www.sigmaaldrich.com/catalog/product/sigald/237213?lang=en&#38;region=US</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">clear boxes with lids</td>
			<td style="width:149px;">Rosti Mepal</td>
			<td style="width:139px;">Rubber maid</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">paper towels</td>
			<td style="width:149px;">In House</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">cell scraper</td>
			<td style="width:149px;">CellTreat</td>
			<td style="width:139px;">229310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PBS</td>
			<td style="width:149px;">In house prepared</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">70% Ethanol</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:139px;">64-17-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">microcentrifuge tubes sterile</td>
			<td style="width:149px;">Santa Cruz</td>
			<td style="width:139px;">sc-200273</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">biohazard bags with holder (desktop)</td>
			<td style="width:149px;">Heathrow Scientific</td>
			<td style="width:139px;">HS10320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Nitrile Gloves</td>
			<td style="width:149px;">VWR</td>
			<td style="width:139px;">89428</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">oxygen electrode</td>
			<td style="width:149px;">eDAQ</td>
			<td style="width:139px;">EPO354</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">pH meter</td>
			<td style="width:149px;">Jenway</td>
			<td style="width:139px;">3510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">pH paper/ pHydrion</td>
			<td style="width:149px;">Sigma Aldich</td>
			<td>Z111813</td>
			<td></td>
		</tr>
	</tbody>
</table>

anaerobic growth and maintenance of mammalian cell lines

En mukozal yüzeyler tümörler ve kök hücre nişler degradelerin yanı sıra, anoksik vücudun coğrafi alanlardır. Önceki çalışmalar gösteriyor ki kuluçka normoxic (%5 CO2 havada) içinde veya hipoksik (düşük oksijen düzeyleri) koşulları takip bir anoksik kuluçka (ücretsiz oksijen yokluğu) sonuçlarına göre sınırlı canlılık (2-3 gün). Roman bir metodoloji sağlayan bir anoksik hücre ekimi (en az 17 gün; en uzun süre test için) geliştirilmiştir. En önemli bir özelliği Bu metodoloji, oksijensiz sisteme giriliyor sağlamaktır. Bu medya ve reçeteye göre sarf tüpler, yemekler, şişeler ve pipetler bir anaerobik gaz karışımı (H2, CO2, N2) ile malzeme anoksik (oksijen olmayan) ortamına equilibrate izin vererek takip gaz giderme tarafından elde edilir kullanım öncesinde. Ne zaman elde photomicrographs elde edilen filmler eserler içermediğinden emin olmak için ek bakım uygulanmalıdır. Oksijen yokluğunda, hücre morfolojisi önemli ölçüde değişmiş. İki farklı morphotypes tüm hücreler için anaerobik yetiştirilen belirtilmiştir. Yeteneği büyümeye ve oksijen yokluğu memeli hücreleri korumak için Hücre fizyolojisi, polymicrobial etkileşimleri ve Roman kanser ilaç geliştirme biyosentetik yollar karakterizasyonu analizlerine uygulanabilir.

Bu iletişim kuralı gücünü verdiği destek, uzun ömürlü ve birden çok hücre satırı büyüme ve bir derin değiştirme ve ıraksak evrim süreçleri sonucu hücre morfolojisi25vardır tanıma yatıyor. Bu çalışmanın en önemli unsuru transfer ve sıkı anaerobiosis altında hücrelerin bakımı sağlamaktır. Bu bir anaerobik odası (Şekil 1) iletişim kuralının en üst düzeye çıkarmak için düzenlenen ve hücreler...

Watch this Scientific Journal Video about Anaerobic Growth and Maintenance of Mammalian Cell Lines at JoVE.com

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Anaerobik büyüme ve bakım memeli hücre hatları

Burada, kurulan hücre hatları anaerobik uzun vadeli ekimi sağlar yeni bir yöntem açıklanmaktadır. Test edildi en fazla yaşam süresi 17 gün var. Bu yöntem sitotoksik ajanlar test ve anoxically hücreleri çoğaltma Fizyoloji keşfetmek için uygundur.

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...

anaerobic-growth-and-maintenance-of-mammalian-cell-lines

Research

JoVE Journal

Biology

10.0K Görüntüleme.  Midwestern University. Burada, kurulan hücre hatları anaerobik uzun vadeli ekimi sağlar yeni bir yöntem açıklanmaktadır. Test edildi en fazla yaşam süresi 17 gün var. Bu yöntem sitotoksik ajanlar test ve anoxically hücreleri çoğaltma Fizyoloji keşfetmek için uygundur.

Video: Anaerobic Growth and Maintenance of Mammalian Cell Lines

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

Tripsin ile Pasajlanması tonlar İnsan Embriyonik Kök Hücre hatları

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

Biyoloji

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

Geniş Alan Mikroskop kullanarak Uzun vadeli Görüntüleme Memeli Hücreleri

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Video Biyoinformatik İnsan Embriyonik Kök Hücre Colony Büyüme Analizi

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

Memeli Hücreleri RNAi Tarama MİSYON esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Memeli Hücreler, Hücre Döngüsü Pozisyon Analizi

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

Moxi Z Mini Otomatik Hücre Sayacı Kullanımı Memeli Hücre Sayımı, Hücre Boyutu ve Hücre Sağlık Belirlenmesi

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

İnsan Pluripotent için alternatif Kültürleri Hücre Üretim, Bakım ve Genetik Analizi Kök

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Memeli Hücrelerde Heme Sentezi Düzeylerinin Ölçümü

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

CRISPR / Cas9 üzerinden Memeli Hücre Hatlarında Genomik Delesyonlar Üretimi

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

Bir bakımı<em&gt; Drosophila melanogaster</em&gt; Nüfus Kafes

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...