Dr. Minghan Tong ve S. Kato yazarlar nazikçe sağlayan reaktifler ve fareler için teşekkür ederiz. Biz yararlı tartışmalar Zou laboratuvar üyeleri teşekkür ederim. Bu eser kısmen hibe tarafından desteklenmiştir 973 Çin Bakanlığı bilim ve Teknoloji (çoğu) [2014CB964704 2015CB964503], 9 insanların Hastanesi, Shanghai Jiao Tong Üniversitesi Tıp Fakültesi klinik araştırma programı Program. Hücre biyolojisi ve çekirdek tesis Kimya Biyoloji, moleküler hücre bilim, Shanghai Enstitüsü biyokimyası ve hücre biyolojisi, Çin Bilimler Akademisi mükemmelliği CAS merkezi çekirdek tesis yardım için teşekkür ederiz.

Hayvanlarla ilgili tüm yordamlar Stanford idari Panel üzerinde laboratuvar hayvan bakım (APLAC) tarafından onaylanmış protokolüne göre gerçekleştirilen ve hayvan bakım ve kullanım Komitesi Shanghai Enstitüsü Biyokimya ve hücre tarafından kabul edildi Biyoloji.
1. hazırlık
<ol><li>Osteoklast belirli Raptor silme fareler (Raptorfl/fl; oluşturmak Ctsk-cre, ahiret CtskRap) Ctsk-cre fareler ile Raptorfl/fl fareler çiftleşme tarafından. Bu çalışmada WT denetimi olarak Raptorfl/fl fare kullanın.</li>
	<li>Bir konteyner ile izole kemik tutmak için buz var.</li>
	<li>
Kültür ortamı hazırlamak.
	<ol>
<li>α-MEM kültür orta minimum temel orta alfa (α-MEM) 1 x glutamin, penisilin-streptomisin ve % 10 fetal buzağı serum ile ilave olarak hazırlayın.</li>
		<li>α-MEM orta ve M-CSF 50 mL 20 ng/mL oluşan kemik iliği makrofaj indüksiyon orta hazırlayın.</li>
		<li>M-CSF ve RANKL 20 ng/mL sırasıyla, α-MEM orta 50 mL oluşan Osteoklast indüksiyon orta hazırlayın.</li>
	</ol>
</li>
	<li>
Aşağıdaki arabellekleri tuzak boyama önce hazırlayın.
	<ol>
<li>Aseton, 2.5 mL sitrat çözüm ve 0.8 mL % 37 (vol/vol) formaldehit çözeltisi mL 6.5 oluşan düzeltme çözüm hazırlamak.</li>
		<li>Çözüm boyama tuzak hazırlayın.<ol>
<li>Deiyonize su ile 37 ° c prewarm</li>
			<li>100 µL hızlı garnet GBC temel çözüm ve sodyum nitrit çözeltinin 100 µL 1.5 mL microtube ve 30 s. bırakmak için nazik INVERSION tarafından 2 min için stand karışımı karıştırın.</li>
			<li>Prewarm 9 mL deiyonize su ile 37 ° c Diazotized hızlı garnet GBC temel çözüm 200 µL adım 1.4.2.2, 100 µL naftol AS-BI fosfat çözüm, 400 µL asetat çözüm ve 200 µL tartarat çözüm ekleyin.</li>
			<li>Bir su banyosu 37 ° C'de çözümde karışım boyama tuzak tutmak</li>
		</ol>
</li>
	</ol>
</li>
	<li>
TUZAK etkinlik miktar kültür ortamının önce aşağıdaki arabellek hazırlayın.
	<ol>
<li>Tartarat arabellek (50 mL) hazırlamak: 0,33 M L tartarik asit, 0,33 M sodyum tartarat dibasic 48 mL 2 mL kurutmak. 4,9 pH değerine ayarlayın.</li>
		<li>4-Nitrophenyl fosfat disodyum (pNPP) arabellek (200 mL) hazırlamak: 1.502 g glisin, 41 mg MgCl2, ZnCl227.2 mg ve 180 mL deiyonize su. 10,4 ile 3 M NaOH pH değerine ve 200 mL deiyonize su ile birime ayarlayın.</li>
		<li>PNPP arabellek fosfataz substrat ile (bir 96-şey plaka) hazırlamak: 49.37 mg fosfataz substrat ve pNPP arabelleği adım 1.5.2 175 µL.</li>
		<li>Substrat arabellek (9 mL/96-iyi plaka) hazırlamak: 6,24 mL deiyonize su, asetat çözüm 360 μL ve tartarat arabellek 2.4 mL. Gönderen Vortex mix ve kullanımdan önce 37 ° C'de ısıtın.</li>
		<li>9 ml substrat karışımı ve 10 için girdap yapmak için Önceden ısıtılmış substrat buffer(1.5.4) fosfataz substrate(1.5.3) ile pNPP tamponunun 90 μL eklemek s.</li>
	</ol>
</li>
</ol>2. diseksiyon (gün -1)
<ol><li>3 aylık bir kadın WT ve RapCtsk fareler CO2 tarafından ayrı ayrı ötenazi. CO2 inhalasyon eğitimli personel tarafından uygun ekipman ile gerçekleştirin.</li>
	<li>Bir ölçek ile % 75 etanol (vol/vol) bakteriyel kontaminasyonu önlemek ve daha sonra sırtüstü pozisyonda bir diseksiyon tahtada yer 5 min için 100 mL 6 fare bırakın. Bir kesik dikey olarak tibia distal yapmak ve arka bacak boyunca cilt oftalmik makasla incelemek.</li>
	<li>Kalça eklemi eklem bağ makasla kesme ve hind bacaklarda bagajdan yerinden çıkar.</li>
	<li>Diz eklem eklem bağ kesilmiş ve dikkatle diz eklemi üzerinden femur ve tibia ilişkisini kaldır. Yavaşça yumuşak doku makasla çıkarın.</li>
	<li>Bütün kemikleri tek bir fare bir genotip içinde altı iyi bir tabak her şey α-MEM buz üzerinde 2 mL ile yerleştirin. Not: hücre canlılığı korumak için kemik az 1 h için bu koşullarda tutulmalıdır.</li>
</ol>3. yalıtım (gün -1)
<ol><li>Bir altı-şey plaka % 75 etanol (3 mL) ile bir kuyu ve diğer 5 kuyuları α-MEM orta (3 mL) ile doldurun.</li>
	<li>5 kere α-MEM orta 10 ile 15 s ve yıkama kemikler için etanol yıkama bir genotip tüm kemikleri koymak lar her zaman.</li>
	<li>Epifizleri makas ile kesti ve kemik boşluğu ve floş iliğini α-MEM orta içine 50 mL santrifüj tüpü ile 0,45 mm şırınga iğne yerleştirin.</li>
	<li>Kemik diğer ucundan aynı yöntemi kullanarak kemik boşluğu floş. En az iki kez kemik soluk olana kemik boşluğu iyice yıkamak için yineleyin.</li>
	<li>Bir hücre Pelet vasıl 800 x g ve 4 ° C 5 dk. aspiratı süpernatant elde etmek için kemik iliği santrifüj kapasitesi.</li>
	<li>Kırmızı kan hücre lizis arabellek 3 mL santrifüj tüpü ve pipet yavaşça kemik iliği resuspend ekleyin. Santrifüj tüpü için kırmızı kan hücreleri parçalayıcı için 8 dakika buzda tut.</li>
	<li>α-MEM orta 6 mL hücre lizis durdurmak için santrifüj tüpüne ekleyin. 500 x g ve 4 ° C'de 10 dakika santrifüj kapasitesi ve süpernatant Aspire edin.</li>
	<li>3 mL α-MEM kültür orta resuspend ve hücreleri bir altı-şey plaka (genellikle bir fare iyi başına) içine yerleştirin.</li>
	<li>%5 CO2 kuluçka 37 ° C'de gecede kuluçkaya.</li>
</ol>4. kaplama (0 gün)
<ol><li>Süpernatant ertesi sabah ekli olmayan hücreleri toplamak için 15 mL santrifüj tüpü aktarın.</li>
	<li>800 g ve 5 min için 4 ° C x santrifüj kapasitesi ve süpernatant Aspire edin.</li>
	<li>4 mL α-MEM kültür orta resuspend.</li>
	<li>Hücre çözümü 20 µL alıp Trypan mavi 20 µL ile karıştırın. Karışımı 10 µL sayım odasına ekleyin ve çözüm mL başına hücre sayısı elde edilir.</li>
	<li>Uygun bir birim olarak 500.000 hücre/mL hücre çözümü elde etmek için α-MEM kültür ortamının ekleyin.</li>
	<li>500 µL ve kemik iliği makrofaj indüksiyon orta 50 µL 24-şey plaka ve 96-şey plaka, her kuyuya sırasıyla ekleyin.</li>
	<li>500 µL ve hücre çözümü 50 µL 24-şey plaka ve 96-şey plaka, her kuyuya sırasıyla ekleyin.</li>
	<li>%5 CO2 kuluçka 37 ° C'de 3 gün kuluçkaya.</li>
</ol>5. farklılaşma (3-7 gün)
<ol><li>Üç gün sonra kaplama, 96-iyi-plaka her kuyudan 50 µL orta toplamak ve -20 ° C'de dondurmak ve kalan orta Aspire edin.</li>
	<li>1 mL ve 100 µL Osteoklast indüksiyon orta 24-şey plaka ve 96-şey plaka her kuyuya sırasıyla ekleyin.</li>
	<li>37 ° C de 2 gün kuluçkaya.</li>
	<li>Orta 50 µL her kuyudan toplamak ve kalan orta Aspire edin.</li>
	<li>Osteoklast indüksiyon orta her kuyunun içine ekleyin.</li>
	<li>37 ° C'de 1 gün için kuluçkaya. Not: Bu zaman noktada, ters bir mikroskop altında (şekil 2A) büyük, multinucleated Osteoklastlar görülmelidir.</li>
	<li>Osteoklastlar görülmez, Osteoklast indüksiyon orta değiştirmek ve Osteoklastlar görülmektedir kadar her gün gözlemlemek.</li>
	<li>Osteoklastlar oluşmuş sonra orta 50 µL 96-şey plaka her kuyudan toplamak.</li>
</ol>6. tartarat dayanıklı asit fosfataz (tuzak) Boyama
Not: Olgun Osteoklastlar vitro kültür (Adım 4) 6-7 gün sonra mevcut olabilir.
<ol><li>Orta Aspire edin ve yavaşça 3 kez 1 x PBS ile yıkayın.</li>
	<li>Hücreleri 96-şey plaka her şey 100 µL 30 için düzeltme çözümü ile fix s, oda sıcaklığında.</li>
	<li>Fiksasyon sonra düzeltme çözüm Aspire edin ve yavaşça 37 ° C'ye prewarmed deiyonize su ile 3 kez yıkamak Deiyonize su Aspire edin.</li>
	<li>TUZAK leke 96-şey plaka her kuyuya 100 µL ekleyin ve kuluçka 37 ° C'de 30 dk ve ışık kalkan plaka yer.</li>
	<li>Boyama sonra tuzak leke Aspire edin ve yavaşça 3 kez deiyonize su ile yıkayın.</li>
	<li>40 X büyütme oranında ters bir mikroskop kullanarak görüntü Osteoklastlar.</li>
</ol>7. tuzak etkinlik Quantification kültür ortamının
<ol><li>30 µL kültür süpernatant her şey 96-şey plaka adım 5.1, 5.4 ve 5,8 toplamak; Kültürlü Osteoklast indüksiyon orta 30 µL negatif kontrol kullanın. Örnekleri yeni bir 96-şey tabağına yerleştirin.</li>
	<li>Substrat mixture(1.5.5) 90 μL 96-şey plaka her kuyunun içine ekleyin.</li>
	<li>Plaka 37 ° C'de bir kuluçka 1 h ve ışık kalkan için yerleştirin.</li>
	<li>Reaksiyon 96-şey plaka her kuyuya 50 µL 3 M NaOH ekleyerek durdurmak.</li>
	<li>Ölçmek theabsorbance, dalga boyu 405 nm.</li>
</ol>8. Batı kurutma
Not: vitro kültür 24-şey plaka 6-7 gün sonra olgun Osteoklastlar görünür olmalıdır.
<ol><li>Orta Aspire edin.</li>
	<li>Yavaşça önceden soğutulmuş 1 x PBS 3 kere yıkayın.</li>
	<li>PBS Aspire edin.</li>
	<li>1 SDS lizis arabellek içeren proteaz inhibitörü kokteyl x 100 µL ekleyin.</li>
	<li>Isı lysates 105 ° c 10 dakika ve 10 dakika süreyle santrifüj 12.000 × g ve 4 ° C protein içeren supernatants toplamak.</li>
	<li>% 10 SDS-sayfa11 tarafından 30 µg protein içeren lysates ayırın ve sonra bir polivinilidin florid (PVDF) membran aktarın.</li>
	<li>%5 30 dk için yağsız süt ile membranlar engelleyin.</li>
	<li>Membranların Anti-Raptor, anti-P-S6, anti-S6, anti-β-aktin, gecede 1:1,000 seyreltme 4 ° C'de % 5 yağsız süt ile kuluçkaya.</li>
	<li>Tris tamponlu tuz ile ara-20 (TBST) ile membranlar 10 dakika oda sıcaklığında yıkayın.</li>
	<li>Adım 8,9 iki kez tekrarlayın.</li>
	<li>Membranlar horseradish peroksidaz (HRP) ile kuluçkaya-Anti-fare ya da tavşan IgG antikorlar 1:5,000 seyreltme oda sıcaklığında 1 h için %5 yağsız süt, Birleşik.</li>
	<li>Membranlar 3 kez ile TBST 10 dk için oda sıcaklığında yıkayın.</li>
	<li>Chemiluminescent algılama için Batı chemiluminescent HRP substrat membranlar ekleyin ve oda sıcaklığında 5 min için kuluçkaya. Fazla yüzey drenaj ve röntgen film açıkları ortaya çıkarmak.</li>
</ol>

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Tüm yazarlar hiçbir çıkar çatışmaları ellerinde devlet.

Osteoclastogenic tahlil yalıtmak ve Osteoklastlar vitro12,13kültür için en çok kullanılan yöntemdir. Ise birkaç RANKL tabanlı Osteoklast indüksiyon açıklanan13,14,15olabilirdi, bu da çalışmanın bir iletişim kuralı önceki yöntemleri üzerinde temel bazı değişikliklerle nitelendirdi.
Önceki çalışmada, BMMs hem...

Kemik değişen bir organdır ve dokusunu ve hayatı boyunca Osteoklastlar tarafından yenilenmiş. Osteoklastlar mineralize matris rezorpsiyonu için sorumlu ve dokusunu sentez ve yeni kemik matrisler1salgılar. Kemik rezorpsiyonu ve kemik oluşumu arasındaki dengeyi kemik bakım da dahil olmak üzere Kemik sağlığı için önemlidir kütle ve stimülasyon ve yaralanma yanıt. Bu denge bozulur, bir dizi metabolik kemik hastalıkları, Osteoporoz ve periodontal hastalıkları da dahil olmak üzere meydana gelebilir. Bu hastalıklar, kemik dokusunu2,3kapasite şekillendirme mediatörlerin kemik rezorpsiyonu kaynaklanan kemik kayıplarının aşıyor. Böylece, osteoporoz gibi iskelet bozuklukları tedavisinde ilaç hedefleri geliştirmek için üretimi ve Biyoloji Osteoklastlar4anlamak önemlidir.
Osteoklastlar benzersiz dev multinucleated hücreleri, veya kemik yüzeye yakın bulunan ve monosit/makrofaj aile1' e ait. Ibbotson K. J. vd. Osteoklast benzeri hücreler vitro ile 1,25-dihidroksi-vitamin D35içeren ortam oluşturmak için bir yöntem bildirdi. Makrofaj koloni uyarıcı faktör (M-CSF) ve reseptör aktivatör nükleer faktör-κ B ligand (RANKL) için tanımlayıcısı Osteoklast oluşumu temel faktörler olarak osteoclastogenesis içinde vitro verimliliğini önemli ölçüde artmıştır 1 , 6 , 7. Kültür Osteoklastlar vitro yeteneği anlayışımız oluşturma ve düzenleme, Osteoklastlar geliştirdi.
Memeli/mekanik hedef iki yapısal ve işlevsel olarak ayrı kompleksleri, yani mTORC1 ve mTORC28,9' rapamycin (mTOR) fonksiyonların. İki çok protein kompleksleri onların farklı bileşenleri ve aşağı akım yüzeylerde nedeniyle birbirinden farklıdır. mTORC2 içerirken, rapamycin-duyarlı arkadaşı mTOR (Rictor)9mTORC1 mTOR (Raptor), benzersiz düzenleyici ilişkili protein içerir. mTORC1 entegre ve hücre büyümesi, yayılmasını önleme ve farklılaşma düzenleyen önemli sinyalleri iletmek. Son zamanlarda, biz bu mTORC1 mTORC1 Osteoklastlar10devre dışı bırakabilirsiniz için Raptor silinmesiyle tarafından katabolik kemik rezorpsiyonu ağ içinde önemli bir rol oynar gösterdi. Ancak, temel mekanizmaları daha fazla çalışma gerektirir. Bu da çalışmanın, Osteoklastlar kemik iliği türevi makrofajlar vahşi-tipi (WT) ve Ctsk Rap fareler (BMMs) oluşturmak ve mTORC1 inactivation Osteoklast üzerindeki etkisini incelemek için osteoclastogenic RANKL tabanlı yöntemi kullanıldı oluşumu.

<table>
	<tbody>
		<tr>
			<td>Raptorfl/fl mice</td>
			<td>The Jackson Laboratory</td>
			<td>013188</td>
			<td></td>
		</tr>
		<tr>
			<td>Ctsk-cre mice</td>
			<td></td>
			<td></td>
			<td>a gift from S. Kato, University of Tokyo, Tokyo, Japan</td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Corning</td>
			<td>10-022-CVR</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Gibico</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin streptomycin</td>
			<td>Gibico</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>BioInd</td>
			<td>04-001-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant mouse M-CSF protein</td>
			<td>R&#38;D</td>
			<td>Q3U4F9</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant mouse RANKL protein</td>
			<td>R&#38;D</td>
			<td>Q3TWY5</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>Beyotime</td>
			<td>C3702</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Sigma-Aldrich</td>
			<td>302643</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate solution</td>
			<td>Sigma-Aldrich</td>
			<td>915</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acid Phosphatase, Leukocyte (TRAP) Kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Garnet GBC Base solution</td>
			<td>Sigma-Aldrich</td>
			<td>3872</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Nitrite Solution</td>
			<td>Sigma-Aldrich</td>
			<td>914</td>
			<td></td>
		</tr>
		<tr>
			<td>Naphthol AS-BI Phosphate Solution</td>
			<td>Sigma-Aldrich</td>
			<td>3871</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetate solution</td>
			<td>Sigma-Aldrich</td>
			<td>3863</td>
			<td></td>
		</tr>
		<tr>
			<td>Tartrate solution</td>
			<td>Sigma-Aldrich</td>
			<td>3873</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline</td>
			<td>Corning</td>
			<td>21-031-CVR</td>
			<td></td>
		</tr>
		<tr>
			<td>L-tartaric acid</td>
			<td>Sigma-Aldrich</td>
			<td>251380</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium tartrate dibasic dehydrate</td>
			<td>Sigma-Aldrich</td>
			<td>s4797</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatase substrate</td>
			<td>Sigma-Aldrich</td>
			<td>P4744</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Raptor</td>
			<td>Cell Signaling Technology</td>
			<td>2280</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-P-ribosomal protein S6 (S235/236)</td>
			<td>Cell Signaling Technology</td>
			<td>2317</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-ribosomal protein S6</td>
			<td>Cell Signaling Technology</td>
			<td>2211</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-&#946;-actin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-130300</td>
			<td></td>
		</tr>
		<tr>
			<td>37% formaldehyde</td>
			<td>Xilong scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>polyvinylidene fluoride (PVDF) membrane</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Western Chemiluminescent HRP Substrate (ECL)</td>
			<td>Millipore</td>
			<td>00000367MSDS</td>
			<td></td>
		</tr>
		<tr>
			<td>IX71</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Envision</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.45-mm Syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mosquito forcep</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a rankl-based osteoclast culture assay of mouse bone marrow to investigate the role of mtorc1 in osteoclast formation

Osteoklastlar vardır benzersiz kemik-resorbing kemik iliği monosit/makrofaj soyundan hücre. Osteoklastlar disfonksiyon kemik metabolik hastalıkları, osteoporoz da dahil olmak üzere bir dizi neden olabilir. Patolojik kemik kayıplarının önlenmesi için ilaç hedefleri geliştirmek için hangi precursors Osteoklastlar ayırt mekanizmaları anlaşılması gerekir. Yeteneği yalıtmak ve çok sayıda Osteoklastlar vitro kültür Osteoklast ayrımında belirli genlere rolünün belirlemede önemlidir. İnactivation rapamycin karmaşık 1 (TORC1) Osteoklastlar içinde memeli/mekanik hedef Osteoklast kaç azaltın ve kemik kütlesi artırmak; Ancak, temel mekanizmaları daha fazla çalışma gerektirir. Bu da çalışmanın, ayırmak ve fare kemik iliği dan Osteoklastlar kültür ve mTORC1 inactivation Osteoklast oluşumu üzerine etkisi çalışmaya RANKL tabanlı bir protokol açıklanmıştır. Bu iletişim kuralı, genellikle bir hafta içinde çok sayıda dev Osteoklastlar, başarıyla sonuçlandı. Raptor silinmesiyle Osteoklast oluşumu Engelli ve o mTORC1 Osteoklast oluşumu için kritik olduğunu belirten salgı tartarat dayanıklı asit fosfataz aktivitesini azalmıştır.

Mevcut iletişim kuralını kullanarak, çok sayıda dev Osteoklastlar gün 6 görüldü; Dev Osteoklastlar görülmez, bir gün daha Osteoklast farklılaşma olabilir (şekil 1) gerekli. Başarılı Osteoklast oluşumu (şekil 2A) Boyama tuzak tarafından doğrulandı. Osteoklastlar 3'ten fazla çekirdeği dev şarap kırmızı/mor hücrelerle vardı. 250'den fazla Osteoklastlar WT BMMs (şekil 4A) ...

Watch this Scientific Journal Video about A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation at JoVE.com

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Bu el yazması bir iletişim kuralına izole etmek ve kültür Osteoklastlar vitro fare kemik iliği ve rapamycin karmaşık 1 Osteoklast oluşumunda memeli/mekanik hedef rolü çalışmaya açıklar.

Bir RANKL tabanlı Osteoklast kültür tahlil, fare mTORC1 Osteoklast oluşumunda rol araştırmak için ilik

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

a-rankl-based-osteoclast-culture-assay-mouse-bone-marrow-to

Research

JoVE Journal

Developmental Biology

12.8K Görüntüleme.  National Clinical Research Center of Stomatology. Bu el yazması bir iletişim kuralına izole etmek ve kültür Osteoklastlar vitro fare kemik iliği ve rapamycin karmaşık 1 Osteoklast oluşumunda memeli/mekanik hedef rolü çalışmaya açıklar.

Video: A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Bir Co-kültür Sistemde nöronal bağlantıları Oluşumunda değerlendirilmesi için bir optogenetic Yaklaşım

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Gelişim Biyolojisi

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Bir Enzimatik Yöntem Kaymaklı Kemik İliği Örneklerinden Mezenkimal Kök Hücreler Kurtarma

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

Konfokal Analizi Kullanımı<em&gt; Xenopus laevis</em&gt; Wnt ve Shh morfogen Gradyanların modülyatorlar Soruşturma

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

İnsan Kemik İliği gelen Yalıtımlı Mesangiogenic Ata Hücreleri (MPCs)

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

Fare Embriyonik metatarsallara Kültürü: endokondral Kemikleşmenin bir Fizyolojik Modeli

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

İlaç Tedavi ve<em&gt; İn Vivo</em&gt; Bir Medaka Balık Osteoporoz Modelinde Osteoblast-Osteoklast Etkileşimler Görüntüleme

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

Detaylı Mekansal İfade Haritalar Dinamik İfade Verilerinin Zamansal Sipariş

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

Fare Osteoklast Öncüleri Notch Sinyal uyarılması

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

5-mC Nokta-Blot Testi Kondrosit Farklılaşmasımın DNA Metilasyon Seviyesinin Nicelendirilmesi<em&gt; In vitro</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Fare Malı İnsani Kemik İliği Mikro Çevrelerinin Biyomühendisliği ve Canlı Görüntüleme ile Görselleştirilmesi

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...