Nitya Jain bir NIH/NIAID kariyer geçiş Ödülü 1K22AI116661-01 tarafından desteklenmiştir.

Tüm hayvan yordamları ve kurumsal hayvan bakım ve kullanım Komitesi (IACUC), Massachusetts General Hospital onayı ile gerçekleştirilmiştir.
Dikkat: Bu protokol bir sınıf 3b lazer (LG3) kullanımını gerektirir. LG3 lazer koruyucu gözlük her zaman bu lazer çalışırken kullanılması gerekir. Yaralanma riskini önlemek için uygun eğitim ve güvenlik yönergelere uyulmalıdır.
1. tasarım ve lazer montajı
<ol><li>Işık yayan diyot (LED) bağlanmak (405 nm, 19,3 mW, 1400 mA, fiber birleştiğinde) LED sürücü üzerinden için LED için gelir 4 pinli M8 bağlantı kablosu bağlı. Dikkatli bir şekilde bağlantı tutturmak için sıkıştırın.</li>
	<li>Optogenetics bağlantı kablosu için LED eklemeden önce bağlayıcı tutun LED kadar açılış orada emin olmak için ışık görünür hiçbir engel vardır. Eğer gerekli herhangi bir engelleri ortadan kaldırmak ve optogenetics bağlantı kablosu ataşını delik üfle.</li>
	<li>Fiber optik kanül kanül bağlayıcı kol bir ucunu ekleyerek optogenetics bağlantı kablosu bağlayın. Bağlayıcı kol diğer ucunu bağlantı kablosu takın.  Güvenli bağlantı ama değil viraj veya fiber optik kanül kırmak için dikkatli olmak için yeterli basınç uygulayın. Not: her ne zaman lazer kullanmak değildir kırılgan fiber optik kanül koruyucu başlığı ile kaplıdır.</li>
	<li>LED sürücü 0 ile 1.2 A. ayarlanmış geçerli sınırı sürekli ayar düğmesi ile sürekli dalga (CW) moduna ayarla</li>
</ol>2. Photoconversion iki nokta üst üste hücrelerinin
Not: Erkek ve dişi fareler intracolonic 405 nm ışık için 1-2 gün sonra kendi Doğum maruz kaldılar ve yaş 1 hafta önce kurban edildi.
<ol><li>Karanlık bir yerde bir elektrik prizine bir erişim ile güvenli. LED sürücü sokunuz. Not: ortam ışığı farelerin uzun bir pozlama istenmeyen bir photoconversion hücrelerinin neden olabilir gibi bu yordamı en az beyaz ışık içinde gerçekleştirilir. Kırmızı ışık alternatif olarak kullanılır. Dikkat: Aşağıdaki adımı bir hayvan anestezik kullanımını gerektirir. Anestezi Yönetimi Sınıf II başlıklı veya başka bir uygun atma sistemi kullanılarak gerçekleşir. İnhalasyon anestezi baş dönmesi, uyuşukluk veya hatta bilinç kaybı neden olabilir.</li>
	<li>4-%5 isoflurane % 100 O2 bir indüksiyon kutusunda maruz fare anestezi. Uygun anestezi derinliği sıkıca arka pençe (cevap yok) pinching tarafından doğrulayın. Anestezi burun konisi, yaklaşık % 2 isoflurane üzerinden korumak. İşleyici isoflurane bir uygun başlık veya anestezik atma aygıt kullanımı ile etkilenmemek.</li>
	<li>İntracolonic maruz imzalat fare scruff fare arkası için 1 el yavaşça işaret parmağı ve başparmak ile kullanarak yerleştirin. Karın ortaya çıkarmak için fareyi çevirin. Fare pembe rengini kaybetmeye başlar yoksa onun solunum hızı yavaş başlar kavrama gevşetin. Fare kuyruğu halka ve küçük parmaklar arasındaki güvenli.</li>
	<li>Diğer elini de koruyucu başlığı kanül kaldırın. Süre paraca desteklemek fare orta hat için paralel kanül uzun ekseninin hafifçe yerleştirin fiber optik kanül anüs yoluyla kolon böylece tüm 5 mm Fiber optik fare içinde. Not: Bu yordam yavaş yavaş ve kolon duvar sorgulamaktı değil özenle yapılır. Kıpır kıpır ve kanül büküm dakika içine kolon ilerleme yardımcı olur.</li>
	<li>Koruyucu gözlük don ve maksimum değerine (sonuna kadar saat yönünde) geçerli lazer artırmak. UV ışık varlığı fare karın yarı saydam bakarak onaylayın. Not: LG3 lazer koruyucu gözlük görünürlüğünü önemli ölçüde azaltır. Var bir meslektaşım (giyiyor koruyucu donanım) gözlük donning ve yardımcı üzerine lazer dönüm.</li>
	<li>2 dk. geri kanül yaklaşık 1 mm toplam lazer ışığı kolona her 30 maruz ışığa maruz luminal alanı en üst düzeye çıkarmak için s. Lazer 2 dk sonunda devre dışı.</li>
	<li>Yavaş yavaş kanül fare kaldırın. Anestezi boş bir sıcak kafeste üzerinden geri aldıktan sonra fare ev kafese geri.</li>
</ol>3. yalıtım bağırsak lenfositler
<ol><li>Hank'in dengeli tuz çözüm/buzağı serum (HBSS/CS) arabelleği 1 L, epitel şerit arabellek 200 mL, 500 mL yıkama medya ve ethylenediaminetetraacetic asit (EDTA) 200 mL hazırlamak-ücretsiz yıkama medya. Not: Reaktif birimleri 9 Örneklem büyüklüğü için hesaplanır.<ol>
<li>HBSS/CS tampon (HBSS; %5 CS) HBSS için 950 mL 50 mL CS ekleyerek getirin. Onu buza saklayın.</li>
		<li>[HBSS; %5 CS; 1 mM dithiothreitol (DTT); 5 mM EDTA; 15 mM 4-(2-Hydroxyethyl) piperazin-1-ethanesulfonic asit (HEPES) (pH 7.2-7,5)] epitel şerit arabellek için 185 mL CS, 1 M DTT, 0,5 M EDTA 2 mL ve 1 M HEPES tampon 3 mL 200 µL 10 mL ekleyerek hazırlamak HBSS. 37 ° C su banyosu epitel şerit arabellekte bir yer.</li>
		<li>(HBSS; % 1 CS; 1 mM EDTA) yıkama medya CS 5 mL ve 1 mL 0.5 M EDTA HBSS 494 mL için ekleyerek getirin. Oda sıcaklığında saklayın.</li>
		<li>EDTA içermeyen yıkama medya (HBSS; % 1 CS; 15 mM HEPES) 195 mL HBSS 2 mL CS ve 3 mL 1 m HEPES ekleyerek hazırlayın. Oda sıcaklığında saklayın.</li>
		<li>Bir FACS tampon yapmak [1 fosfat tamponlu tuz (PBS) x (pH 7.2 =); % 2 CS] CS 10 mL PBS için 490 mL ekleyerek. Onu buza saklayın.</li>
	</ol></li>
	<li>Doku toplama yemek bir 70 µm hücre süzgeç bir 60 x 10 mm Kültür çanak koyarak hazırlamak. Yemek için HBSS/CS tampon (HBSS; 3.1.1. adımda yapılan ve buz üzerinde depolanan % 5 CS arabellek) 10 mL ekleyin ve buz üzerinde yerleştirin.</li>
	<li>Fare ile inhale isoflurane uygun anestezi atma işlemini kullanarak aşırı dozda ötenazi (yaklaşık 3 dk Riziko > % 4 isoflurane Oda hava veya %100 O2). Sonra belirgin nefes kesilmesi ve refleks hareketi, derin ağrı yokluğu sıkıca bir arka pençe pinching tarafından doğrulayın (forseps kullanarak önerilir). Ötenazi ile cerrahi makas ya da yeni bir usturanın kullanarak bir işten çıkarma emin olun. Not: Servikal yerinden çıkması boyutu ve hayvan yaş nedeniyle bu adım için tavsiye edilmez. Bu yöntemler üzerinde ötenazi 2013 travma yönergeleri ile tutarlı olduğundan ve MGH IACUC tarafından onaylandı.</li>
	<li>Karın açmak için temiz forseps ve makas kullanarak kolon hasat. Bağırsak kolon ortaya çıkarmak için fare karın 1 tarafına yansıtır. İlk o (doğru anüs) sadece distal transecting tarafından tüm kolon kaldırmak çekum için. Sonra iki nokta üst üste forseps ile sıkıca açgözlü iken, makas üretra Rectuma kesmek için kullanın. Anüs mümkün olduğunca yakın olarak kolon kesme. Dalak Kolonu çıkardıktan sonra hasat (bkz: 4.1-4.2 aşağıdaki adımlar). Not: photoconversion iletişim kuralı yalnızca ~ % 25 yenidoğan'ın kolon (fiber optik kanül 5 mm) hedefleyen iken, tüm kolon hücre yalıtım modunda yardım için hasat edilir.</li>
	<li>İki nokta üst üste iki nokta üst üste ve kuru kağıt havlu üzerine içeriğini itmek için forseps kullanarak temizleyin. Doku ince bir kültür yemek kapağında bir hamur içine iki nokta üst üste doğrayın ve bu hamur hücre süzgeç Petri kabına aktarmak için jilet kullanarak lunaparkçı.</li>
	<li>Temiz 8 mm uzun manyetik heyecan çubuğu için süzgeç ekleme ve çanak 9-pozisyon manyetik karıştırıcı tabağa yerleştirin. Manyetik heyecan bar hızını ayarlamak için 800 devir/dakika ve oda sıcaklığında 5 min için hücreleri kuluçkaya. Arabellek atın ve 10 mL HBSS/CS (HBSS; 3.1.1. adımda yapılan ve buz üzerinde depolanan % 5 CS arabellek) kullanarak yıkama yineleyin. Arabellek yıkama sonunda atmak.</li>
	<li>Epitel hücreleri prewarmed epitel şerit arabellek (HBSS % 5 CS 1 mM DTT; 5 mM EDTA; 3.1.2 adımda yapılan ve 37 ° C su banyosu içinde depolanmış 15 mM HEPES [pH 7.2-7,5] arabellek) 10 mL ekleyerek hücre süzgeç ayırmak ve 30 dk içinde 3 için 800 rpm'de karıştırın 7 ° C inkübatör. İnkübasyon sonunda, epitel kesir bir temiz 15 mL tüp için zenginleştirilmiş arabellek aktarın. Not: sağlam lamina propria hücrelerde hücre süzgeç sırasında bu adım11kalırken epitel ve ilişkili hücreleri (bağırsak epitel hücreleri ve bağırsak epitel lenfositler) arabelleği serbest.<ol>
<li>Zenginleştirilmiş Bağırsak epitel kesir (IELs) vasıl 700 x g için oda sıcaklığında 5 dk santrifüj kapasitesi. Süpernatant atın ve hücre Pelet FACS arabellek 1 mL resuspend. Kullanılmak üzere adım 3,15 buzda saklayın.</li>
	</ol></li>
	<li>2 hücre süzgeç içinde lamina propria kesir yıkama x HBSS/CS ile. Bunu yapmak için 10 mL HBSS/CS çözeltisi (HBSS; buz üzerinde depolanan adım 3.1.1 yapıldı % 5 CS arabellek) hücre süzgeç ekleyin ve 37 ° C kuluçka 5 min için 800 rpm'de karıştırın. Arabellek her yıkama sonra atın.</li>
	<li>Örnek 2 yıkama yıkama medya x. Yıkama medya (HBSS; % 1 CS; adım 3.1.3 ve oda sıcaklığında depolanmış 1 mM EDTA arabellek) 10 mL hücre süzgeç ekleyin ve 15 dakika 37 ° C kuluçka 800 rpm'de karıştırın. Arabellek her yıkama sonra atın.<ol>
<li>Yıkama medya kullandıktan sonra adım 3.14 kullanımda buzda saklayın.</li>
	</ol></li>
	<li>Örnek 2 yıkama x HBSS ile/CS. 10 mL HBSS/CS tampon (HBSS; buz üzerinde depolanan 3.1.1. adımda yapılan % 5 CS arabellek) hücre süzgeç ekleyin ve 37 ° C kuluçka 5 min için 800 rpm'de karıştırın. Arabellek her yıkama sonra atın.</li>
	<li>EDTA içermeyen yıkama arabellek dokusunda kuluçkaya. 10 mL EDTA içermeyen yıkama arabellek (HBSS; % 1 CS; 3.1.4. adımda yapılan ve oda sıcaklığında depolanmış 15 mM HEPES tampon) hücre süzgeç ekleyin ve karıştırma olmadan oda sıcaklığında 10 dakika plaka kuluçkaya. Bu adımın sonundaki arabellek atmak.</li>
	<li>Örnek 2 yıkama x HBSS/CS ile ve enzim çözüm hazırlamak. HBSS/CS (HBSS; % 5 CS arabellek buz üzerinde depolanan adım 3.1.1 yapılan) 10 mL ekleyin arabellek hücre süzgeç ve 10 dk 37 ° C kuluçka için 800 rpm'de karıştırın. Arabellek her yıkama sonra atın.<ol>
<li>Enzim çözüm hazırlamak (EDTA içermeyen yıkama arabellek; 0.1 mg/mL DNaz ben; 167 mg/mL collagenase) ikinci yıkama süresi boyunca: 9 mg eklemek DNaz ben ve 15 mg liyofilize collagenase 90 mL EDTA ücretsiz için arabellek yıkayın. Çözüm, oda sıcaklığında saklayın.</li>
	</ol></li>
	<li>Lamina propria lenfositler (LPLs) elde etmek için hücre süzgeç bulunan lamina propria kesir sindirmek. Enzim çözüm 10 mL ekleyin (EDTA ücretsiz yıkama arabellek; 0.1 mg/mL DNaz ben; 3.12.1 adımda yapılan ve oda sıcaklığında depolanan 167 mg/mL collagenase arabellek) hücre süzgeç için ve 45 dk 37 ° C kuluçka için 800 rpm'de karıştırın. Not: LPLs sonra enzimatik sindirim çözüm içine salınır.<ol>
<li>Hücreleri içeren enzim çözüm toplamak ve 50 mL konik tüp içine yeni bir hücre süzgeç aracılığıyla filtre.</li>
	</ol></li>
	<li>20 mL soğuk yıkama medya (HBSS; % 1 CS; 1 mM EDTA made ın adım 3.1.3 ve saklı 3.9.1. adımda buzda arabellek) enzim çözüm collagenase faaliyetleri engellemek için filtre uygulanmış hücreleri ekleyin. Tüp vasıl 700 x g oda sıcaklığında 6 dk santrifüj kapasitesi. Süpernatant atmak ve LPLs 1 mL FACS arabellek içeren pelet resuspend.</li>
	<li>IEL (Kimden adım 3.7.1) ve lipoprotein LİPAZ (Kimden adım 3.14) kesirler birleştirir ve onları floresan Birleşik antikorlar akış sitometrik çözümlemesi ile leke (bkz. Adım 5). Not: bölme bilgi isterseniz IEL ve lipoprotein LİPAZ kesirler ayrı ayrı lekeli.</li>
</ol>4. yalıtım lenfositleri dalak üzerinden
<ol><li>Toplama tüp microcentrifuge tüp içine 500 µL FACS arabelleği ekleyerek hazırlayın. Onu buza saklayın.</li>
	<li>İki nokta üst üste (adım 3.4) kaldırma sonra bütün dalak hasat ve toplama tüpü yerleştirin. Doku buz üstünde depolar.</li>
	<li>Bir elektrik doku homogenizer kullanarak, dalak yaklaşık 1 dk. santrifüj microcentrifuge tüp içinde 300 x g [mikro santrifüj] oda sıcaklığında ve aspiratı süpernatant kapalı 5 min için de örnek lunaparkçı.</li>
	<li>Kırmızı kan hücre lizis 500 µL amonyum klorür potasyum (ACK) lizis arabelleği hücre ölümü tarafından ozmotik lizis başlatmak için örnek resuspending tarafından gerçekleştirin. Örnek 1-2 dk oda sıcaklığında kuluçkaya ve sonra FACS arabellek 725 µL içinde resuspend izin.</li>
	<li>300 x g [mikro santrifüj] oda sıcaklığında ve aspiratı süpernatant kapalı 5 min için de örnek santrifüj kapasitesi. Pelet FACS arabellek 500 µL içinde resuspend ve yeni microcentrifuge tüp içine 40 µm hücre süzgeç aracılığıyla filtre. Tüm örnek için yeni bir microcentrifuge tüp aktarmak ve floresan Birleşik antikorlar akış sitometrik çözümlemesi ile leke (bkz. Adım 5).</li>
</ol>5. Dendra-r+ hücreleri kullanarak akış sitometresi tanımlaması
<ol><li>300 x g [mikro santrifüj] oda sıcaklığında ve aspiratı süpernatant kapalı 5 min için de IEL/LPL ve dalak örnekler santrifüj kapasitesi.</li>
	<li>Granül mix boyama antikor 100 µL içinde resuspend. Mix 1 µL fluorescently Birleşik anti-CD45 antikor FACS arabelleği örnek başına 100 µL ekleyerek boyama antikor hazırlayın. Not: Deneme önce akış sitometresi için en uygun konsantrasyonu belirlemek için Doygunluk bağlama oluştuğu kullanılan antikor titre önemlidir.</li>
	<li>Örnekleri (karanlık bir konumda) buz üzerinde 35 dk için kuluçkaya.</li>
	<li>Örnekleri FACS arabellek 300 µL içinde resuspend ve onları vasıl 300 x g [mikro santrifüj] için oda sıcaklığında 5 dk santrifüj kapasitesi. Süpernatant Aspire edin ve örnekleri FACS arabellek 325 µL içinde resuspend. Akış Sitometresi örneği analiz.</li>
</ol>

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Kimlik ve çalışabileceği ve bağırsaktaki microbiota etkilenmiştir hücrelerin karakterizasyonu önemlidir ve mukozal microenvironment bilgilerinden vücudun geri kalanına nasıl geçirilir anlayış kolaylaştıracaktır. Gut ile ilgili hücre geçiş çalışmak için bir yöntem yalıtımı gut ilişkili hücre tarafından evlat edinen bir transfer alıcı fareler onların doku-posta desenlerini belirlemek ve12,13işlev takip gerektirir. Aktarılan beli...

Memeli gastrointestinal sistem ana makine1ile simbiyotik bir ilişki bulunmaktadır bakteri türleri yüzlerce liman. Yerel ortamda mevcut bağışıklık hücreleri ile bu mikropların barışçıl kullanımını zorlayın ve patojen akınları karşı koruyucu bir bariyer oluşturmak. Böylece, bağışıklık hücreleri ve microbiota arasındaki çift yönlü etkileşimler ana bağışıklık sistemi eğitir ve patojenler için bağışıklık reaktivite eşiğini ayarlar komensal bir topluluk kurmak için kritik öneme sahiptir. Mikrobiyal kompozisyon veya dysbiosis, can değişimler bağışıklık homeostazı rahatsız ve huzursuz bağırsak iltihapları2,3 tip 1 diyabet ve IBD gibi hastalıklar immün aracılı önde gelen dizginlemek düzenleyici devreler .
Sonra hemen Doğum sırasında aynı zamanda bağışıklık sistemini kurmak bağırsak mikrobiyal topluluklar başlar benzersiz bir gelişimsel pencere dönemi4olgunlaşır. Postnatal microbiota istikrarlı, vardiyalar halinde doğal olarak meydana gelen topluluk kompozisyon ve sık sık5ile değil. İki farklı anatomik konumda bağırsak - lamina propria ve bağırsak epitel6microbiota ile etkileşim bağışıklık hücreleri bulunur. Çeşitli tiplerde bağışıklık hücreleri lenfositler (T hücreleri, B hücreleri ve doğuştan gelen lenfoid hücre gibi) (hangi katmak dendritik hücreler, monosit ve makrofajlar) myeloid hücrelerin yanı sıra dahil bağırsak, mevcut. Bu hücreler, olarak da bilinen hematopoetik hücreler, çok sayıda bağırsak bariyer korumak ve homeostazı korumak işlevler gerçekleştirin.
Bağırsak siteleri kendi düzenleyici işlevlerine ek olarak, mukoza bağışıklık hücreleri sistemik dokunulmazlık7,8,9düzenleyen mikrobiyal iletileri ekstra bağırsak sitelere de taşıyabilir. Bu araştırma ilgi alanıdır ve onların işlevini prob için bağırsak dokusu dışında geçirmek bağışıklık hücreleri tanımlamak yöntemleri ihtiyacını vurgular. Rapor burada protokol photoconvertible floresan protein etiket hücrelere istismar edilir bir ticari olarak mevcut fare modeli kullanır. PhAMeksize fareler ubiquitously ultraviyole (UV) ışık10tarafından geri dönüşümsüz etkinleştirme üzerine kırmızı floresans için açık yeşil bir floresan Dendra2 protein hızlı. Yeni doğan farelerin kolon 405 nm ışık sağlamak için fiber optik kanül kullanarak, kaynaklı veya kolon aktarıldığı photoconverted hematopoetik hücreler, dalak bulunabilir göstermektedir.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Laser</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Light Emitting Diode (LED)</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">M405FP1</td>
			<td style="width:247px;">CAUTION: this is a Class 3b laser. Safety goggles must be worn when using the laser. It emits a 405 nm wavelength with a current of 1400 mA. It is fiber-coupled. It accepts SMA connector. https://www.thorlabs.com/thorproduct.cfm?partnumber=M405FP1</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">LED driver</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">LEDD1B</td>
			<td style="width:247px;">Drives a constant current of 1200 mA through the laser. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2616</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:259px;">Optogenetics patch cable</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">M87L01</td>
			<td style="width:247px;">1 m long cable with an SMA connector. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=11405&#38;pn=M87L01#11454</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">Fiber optic cannula</td>
			<td style="width:201px;">Doric lenses&#160;</td>
			<td style="width:167px;">MFC_480/500-0.5_5mm_ZF1.25_C45</td>
			<td style="width:247px;">5 mm long cannula with an outer diameter of 500 &#181;m and an inner diameter of 480 &#181;m. The NA value is 0.5. The ferrule is zirconia, 1.25 mm OD. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=6036</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">Power supply</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">KPS101</td>
			<td style="width:247px;">Supplies 15 V with a current of 2.4 A https://www.thorlabs.com/search/thorsearch.cfm?search=KPS101</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:259px;">LG3 laser safety goggles&#160;</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">LG3</td>
			<td style="width:247px;">Orange lenses with 47% visible light transmission https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=762&#38;pn=LG3#2523</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Red light&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>74327-10</td>
			<td style="width:247px;">15 W lamp https://us.vwr.com/store/product/12360027/paterson-safelight-electron-microscopy-sciences</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Intestinal cell isolation</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Isoflurane</td>
			<td style="width:201px;">Patterson Veterinary</td>
			<td style="width:167px;">07-893-1389</td>
			<td style="width:247px;">CAUTION: inhalation of this anesthetic may cause dizziness, drowsiness, or even unconsciousness. This anesthetic should be used in a Class II hood.&#160; https://www.pattersonvet.com/Supplies/ProductFamilyDetails/PIF_762328?carouselPageNumber=3</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">1X HBSS</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">14025076</td>
			<td style="width:247px;">Ca/Mg free https://www.fishersci.com/shop/products/gibco-hbss-calcium-magnesium-no-phenol-red-4/14025076?searchHijack=true&#38;searchTerm=14025076&#38;searchType=RAPID&#38;matchedCatNo=14025076</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Calf Serum</td>
			<td style="width:201px;">Hyclone AZM</td>
			<td style="width:167px;">197696</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">EDTA</td>
			<td style="width:201px;">Invitrogen</td>
			<td style="width:167px;">15575020</td>
			<td style="width:247px;">0.5 M concentration https://www.thermofisher.com/order/catalog/product/15575020?SID=srch-srp-15575020</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:259px;">DTT</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:167px;">10197777001</td>
			<td style="width:247px;">CAUTION: harmful if swallowed and causes skin irritation. 1 M concentration https://www.sigmaaldrich.com/catalog/product/roche/dttro?lang=en&#38;region=US</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">HEPES</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">15630080</td>
			<td style="width:247px;">1 M concentration https://www.thermofisher.com/order/catalog/product/15630080?SID=srch-hj-15630080</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:259px;">Petri dish</td>
			<td style="width:201px;">Corning</td>
			<td style="width:167px;">353004</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-easy-grip-tissue-culture-dishes-2/08772f?searchHijack=true&#38;searchTerm=08772F&#38;searchType=RAPID&#38;matchedCatNo=08772F</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">70 micron cell strainer</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">352350</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087712</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Micro magnetic stir bar</td>
			<td style="width:201px;">Fisherbrand</td>
			<td style="width:167px;">1451364</td>
			<td style="width:247px;">Rinse in 70% ethanol after each use. Rinse several times in distilled water prior to each use. The bar is 8 mm long with an octagonal shape. https://www.fishersci.com/shop/products/fisherbrand-octagonal-magnetic-stir-bars-12/1451364#?keyword=1451364</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Magnetic stir plate</td>
			<td style="width:201px;">Corning Laboratory Stirrers</td>
			<td style="width:167px;">440826</td>
			<td style="width:247px;">https://www.coleparmer.com/i/corning-440826-nine-position-stirrer-120-vac-60-hz/8430420?PubID=UX&#38;persist=true&#38;ip=no&#38;gclid=CjwKCAiAqbvTBRAPEiwANEkyCLPLrWABXmOUI0QE53NLV0Owxlcs2V1K6rWbRPOwlcVVDq000FBiQxoCqQAQAvD_BwE</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">Collagenase</td>
			<td style="width:201px;">Roche</td>
			<td style="width:167px;">5401020001</td>
			<td style="width:247px;">https://www.sigmaaldrich.com/catalog/product/roche/05401020001?lang=en&#38;region=US&#38;gclid=CjwKCAiAjuPRBRBxEiwAeQ2QPhE44qlvxjmo1PYu3zCas3w-_d6P9gKjXW82-c1EOm6NjPHCc5WuixoC_0IQAvD_BwE</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">DNase I</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:167px;">10104159001</td>
			<td style="width:247px;">https://www.sigmaaldrich.com/catalog/product/roche/10104159001?lang=en&#38;region=US</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">1X PBS</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">20012-027</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/20012027?SID=srch-hj-20012-027</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">Pipet aid</td>
			<td style="width:201px;">Thermo Scientific</td>
			<td style="width:167px;">14387165</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/s1-pipette-fillers/14387165#?keyword=14387165</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">10 mL serological pipet</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">357530</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">25 mL serological pipet</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">357515</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">15 mL conical centrifuge tube</td>
			<td style="width:201px;">Thermo Scientific</td>
			<td style="width:167px;">339651</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/339650</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">50 mL conical centrifuge tube</td>
			<td style="width:201px;">Thermo Scientific</td>
			<td style="width:167px;">339653</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/339650</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Single cell suspension</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">Eppendorf tubes</td>
			<td style="width:201px;">Seal-Rite</td>
			<td style="width:167px;">1615-5500</td>
			<td style="width:247px;">Holds 1.5 mL. https://www.usascientific.com/Seal-Rite-1.5-ml-tube.aspx</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:259px;">Tissue homogenizer&#160;</td>
			<td style="width:201px;">Kimble</td>
			<td>K7495400000</td>
			<td style="width:247px;">Requires 2 AA batteries. https://www.fishersci.com/shop/products/kontes-pellet-pestle-cordless-motor-cordless-motor/k7495400000</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:259px;">Homogenizer tips&#160;</td>
			<td style="width:201px;">Kimble</td>
			<td style="width:167px;">7495210590</td>
			<td style="width:247px;">Plastic, 0.5 mL tips https://www.fishersci.com/shop/products/kimble-chase-kontes-pellet-pestle-14/k7495210590#?keyword=7495210590</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">ACK lysing buffer</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">A10492-01</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/A1049201?SID=srch-hj-A10492-01</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">40 micron cell strainer</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">08-771-1</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087711</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Antibodies</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">BV786 anti-mouse CD45&#160;</td>
			<td style="width:201px;">BD</td>
			<td style="width:167px;">564225</td>
			<td style="width:247px;">Clone 3O-F11 https://www.bdbiosciences.com/us/reagents/research/antibodies-buffers/immunology-reagents/anti-mouse-antibodies/cell-surface-antigens/bv786-rat-anti-mouse-cd45-30-f11/p/564225</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Live/Dead</td>
			<td>Invitrogen</td>
			<td>L34962</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/L34962</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Other</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Razor blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td style="width:247px;">Use for decapitation. https://us.vwr.com/store/product/4548306/vwr-razor-blades</td>
		</tr>
	</tbody>
</table>

in vivo photolabeling of cells in the colon to assess migratory potential of hematopoietic cells in neonatal mice

Enterik bakteri toplulukları erken yaşta kurulur ve bağışıklık hücre gelişimi ve işlevini etkiler. Yenidoğan microbiota antibiyotik kullanımı ve diyet, otoimmün ve inflamatuvar hastalıklara yatkınlık etkileri de dahil olmak üzere çok sayıda dış etkilere maruz kalabilir. Bozuklukları inflamatuar bağırsak hastalığı (IBD) gibi bağırsak için bağışıklık hücreleri büyük akını ile karakterizedir. Ancak, bağışıklık hücreleri tarafından microbiota şartına ayrıca bağışıklık yanıtı ekstra bağırsak sitelerdeki etkilemek için bağırsaklar dışarı göç. Böylece, tanımlamak ve bağırsak mikrobiyal iletilerden distal sitelere taşıyabilir hücreleri ayırdetmek için gerek yoktur. Burada, ekstra bağırsak sitelerde kimlik geçişten sonra sağlar yeni doğan fareler vivo içinde kolon etiket hücrelere bir yöntem açıklanmaktadır.

Bir fiber optik kablo 405 nm ışık iki nokta üst üsteeksize 2 - gün eski PhAM farelerin teslim etmek için kullanıldı. Önceki deneyler, 30 s maruz kalma en az sitotoksisite (Şekil 1A) ile iki nokta üst üste hücre maksimal photoconversion vermek tespit edilmiştir. Bu nedenle, sıralı 30 s Etkilenmeler kolon farklı kesimlerinin iletişim kuralında tanımlandığı gibi yapılmıştır. Lazer pozlama, fareler hemen eut...

Watch this Scientific Journal Video about In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice at JoVE.com

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Burada açıklanan protokol photolabeling yaklaşım yeni doğan farelerde özellikle kolon ekstra bağırsak sitelere göç bağışıklık hücreleri tanımlamak için kullanır. Bu stratejinin ana bilgisayar-microbiome erken hayat etkileşimlerde eğitim yararlı olacaktır.

Vivo Photolabeling hücrelerinin yenidoğan farelerde hematopoetik hücrelerin göç potansiyeli değerlendirmek için iki nokta üst üste

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

in-vivo-photolabeling-cells-colon-to-assess-migratory-potential

Research

JoVE Journal

Medicine

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

5.7K Görüntüleme.  Massachusetts General Hospital. Burada açıklanan protokol photolabeling yaklaşım yeni doğan farelerde özellikle kolon ekstra bağırsak sitelere göç bağışıklık hücreleri tanımlamak için kullanır. Bu stratejinin ana bilgisayar-microbiome erken hayat etkileşimlerde eğitim yararlı olacaktır.

Video: In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

Ortak Bozuklukları de Novo Mutasyonlar, otizm ve şizofreni gibi tanımlayın Bir Strateji

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

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Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

C57BL/6J Fare Stromal Cells ve Karsinomu Hücreler Meme Nakli

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

Hücre Takip için vivo 19 F MRG

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Farelerde Taksanlar Farmakokinetik ve Farmakodinamik Analiz Karotis Arter İnfüzyonlar

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Rahim içi Telemetri Fare Kontraktil Basıncını Ölçmek<em&gt; İn Vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Atçılık tendinopatisinde Kemik İliği Mezenkimal Kök Hücreler Etiketli Teknesyum- 99m Vivo Görüntüleme ve Takip

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pankreas Kanseri Yeni Tedavi Stratejileri Geliştirme Hücre Özellikleri Kök incelenmesi

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

Singenik Fare Melanom Hücreleri Enjeksiyon Akciğerler Onların Metastatik Potansiyeli belirleme

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

İnsan Tümör ksenogratflarının gelen Fare Hücrelerinin tükenmesi Anlamlı Hedef Hücre Alt Analizi geliştirir

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Vitro ve In Vivo insan hücreleri, C. Elegansve fareler Mitophagy tespiti

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...