This work was supported by the University of Pennsylvania Center for Musculoskeletal Disorders (5 P30 AR050950-09), a grant from the AO Foundation (S-16-12A), the Philadelphia VA Medical Center Translational Musculoskeletal Research Center, and an intramural orthopaedic surgery departmental research development fund. JWA is supported by the University of Pennsylvania Postdoctoral Opportunities in Research and Teaching (PENN-PORT) fellowship funded by the National Institute of General Medical Sciences Institutional Research and Career Development Award (IRACDA; 5 K12 GM081259-08).

척추 동물과 관련된 모든 연구는 펜실베니아 기관 동물 관리 및 사용위원회의 대학 (IACUC)에 의해 승인 된 프로토콜에 따라 수행 하였다. 1. 문화 미디어 준비 <ol><li> 100 ml의 열, 900 ML의 H 2 O의 최소 필수 배지 (MEM) 분말 1.9 g의 탄산 수소 나트륨을 용해하여 α-MEM을 준비하고 2 mM L- 글루타민, 100 단위 / ㎖ 페니실린, 100 μg의 / ㎖ 스트렙토 마이신과 보완 불 활성화 된 소 태아 혈청. 0.22 ㎛의 폴리 에테르 설폰 (PES) 필터를 이용하여 멸균. </li><li> 35 NG로 α-MEM을 보충하여 식세포 매체를 준비 / ml의 재조합 마우스 대 식세포 콜로니 자극 인자 (M-CSF) </li><li> 100 NG / ㎖ RANKL과 식세포 매체를 보충하여 파골 세포 배지를 준비 </li></ol> 2. 골수 세포 분리 <ol><li> 10 ㎖의 α-MEM와 마우스 당 25⅝ 게이지 바늘 한 10 ML의 주사기를 입력합니다. </li&gt; <li> 1.3 L / min의 유량으로 CO 2 10 분간 노출을 통해 마우스 안락사. 해부를 진행하기 전에 자궁 경부 전위와 CO 2 노출에 따라 동물의 죽음을 확인합니다. </li><li> 깨끗한 기술을 사용하여, 해부 및 경골과 대퇴골을 분리합니다. 경골과 대퇴골을 노출 각 뒷다리의 앞쪽 표면을 따라 절개를하기 전에 70 % 에탄올로 마우스 피부를 소독. <ol><li> 대퇴골의 말단을 무료로 무릎 관절을 잘라, 가능한 한 골반에 가까운 대퇴골을 잘라. 대퇴골를 제거하고 최대한 부드러운 조직을 청소합니다. 발목을 절단 경골을 제거하고 가능한 한 많은 연부 조직을 제거합니다. </li></ol></li><li> 2.5 ml의 실내 온도 α-MEM은 하나의 튜브에 하나의 마우스에서 골수 플러시 조합과 15 ML 원뿔 관에 집게와 플러시 골수와 뼈를 잡습니다. 참고 : 성공적인 골수 플러시 t의 착색이 변경됩니다그는 빨간색에서 뼈 베이지합니다. </li><li> 파편은 튜브의 바닥에 정착 이물질 전송을 방지하기 위해 깨끗한 15 ML 원뿔 튜브 돌보는에 뜨는을 전송 할 수 있습니다. 참고 : 일부 세포는 골수 파편으로 정착 할 수 있습니다. 셀들의 많은 수는 필요한 경우 골 플러시 깨끗한 15ml를 튜브에 70 μm의 세포 여과기를 통해 여과 될 수있다. </li><li> 실온에서 5 분 300 XG에 원심 분리기 튜브 (들) 세포 펠렛합니다. </li><li> 진공 용균 완충액 0.5 ml의 암모늄 염화 칼륨 (ACK)에 재현 탁하고 상등액 세포 펠렛을 대기음 적혈구를 용해시키기 위해 3 분 동안 37 ℃에서 배양한다. </li><li> 5 분 동안 300 XG에 솔루션 및 원심 분리기 세포 ACK로 직접 10 ml의 PBS를 추가합니다. 주 : 이전에 붉은 세포 펠렛은 이제 베이지 색이어야한다. </li><li> 100 밀리미터 조직 문화 처리 접시에 진공 흡인 상층 액과에 resuspend 펠렛을 10 ml의 α-MEM, 그리고 판. 가습에 37 ° C에서 하룻밤 세포를 품어조직 문화 인큐베이터. 참고 : 세포를 계산하면이 단계에서 필요하지 않습니다. 이 시간 동안 골수 부착 세포 배양 표면에 부착한다; 비 부착 골수 세포 인구는 현탁액에 남아 있습니다. MCSF이 초기 배양시 배양액에 존재하지 않는다. </li></ol> 파골 세포 전구체 3. 심화 <ol><li> 골수 플러시 한 50㎖에 비 부착 세포를 함유하는 원뿔형 튜브 전사 배양 상등액의 하룻밤 배양 후. 참고 : 골수 중간 엽 전구 세포를 포함 부착 세포와 문화 요리, 폐기 될 수있다 또는 다른 실험을 위해 필요한 경우, 신선한 α-MEM으로 공급. 주 : 파골 세포가 5 일 동안 격일 파골 매체 상쾌한 배지에서 4 × 105 세포 / ㎠의 밀도로 조직 배양 처리 된 요리로 도금하여이 비 부착 골수 세포에서 직접 분화 될 수있다. </l난&gt; <li> 18 ㎖의 최종 부피 비 - 부착 세포 용액 α-MEM을 첨가하고 35 NG / ml의 최종 농도로 M-CSF를 추가한다. </li><li> 6 60mm 현탁 배양 접시에 세포 현탁액 각 3 ㎖를 추가합니다. </li><li> 가습 조직 문화 인큐베이터에서 37 ° C에서 하룻밤 번호판을 품어. 이 시간 동안, M-CSF도 아닌 조직 배양 처리 된 표면에 부착 할 수 대식 세포의 분화를 자극 할 것이다. </li><li> 밤새 3 ㎖ 식세포 신선한 배지로 재 공급 파골 세포 전구체를 배양 후. 이 대 식세포로 분화하지 않은 비 부착 세포를 제거합니다. </li><li> 2 일 또는 문화가 약 70 %의 합류에 도달 할 때까지 CO 2를 37 ° C에서 배양 파골 세포 전구체를 계속 5 %. </li></ol> 4. 파골 세포의 분화 <ol><li> CEL 때까지 실온에서 1 ml의 해양 기원의 단백질 분해 / collagenolytic 효소로 세포를 5 ml의 PBS와 파골 세포 전구체를 씻고 치료LS는 접시의 부드러운 도청에 의해 빠질 수 있습니다. 주 :이 정지 접시에서 배양하는 경우 파골 세포 전구체가 해제 될 수있다; 전구체 해제 할 조직 문화 처리 된 표면에 너무 단단히 연결합니다. 트립신은 파골 세포 전구체를 활성화 할 수 있습니다 피해야한다. </li><li> 50 ML 원뿔 튜브에 각각의 접시 및 전송 세포에 4 ㎖의 α-MEM을 추가합니다. 혈구를 사용하여 세포를 세어 mL를 5 × 104 세포 / 밀도로 세포를 희석시키고 100 NG / ml의 최종 농도로 35 NG / ㎖ 및 RANKL의 최종 농도로 M-CSF를 추가한다. </li><li> 조직 배양 - 처리 된 플레이트에 2.6 × 104 세포 / ㎠의 밀도로 시드 세포는 2 일 동안 가습 된 조직 배양 인큐베이터에서 37 ℃에서 배양한다. 분화는 전형적으로 5 × 104 세포를 각 웰에 시딩 24- 웰 플레이트에서 수행된다. </li><li> 이일 1 ml의 신선한 osteoc와 매체를 대체하여 분화, 재 공급 셀의 시작 후마지막 매체. 참고 : 파골 세포의 분화는 일반적으로 3 일내 완료됩니다. 결과 파골 세포는 TRAP 염색 쉽게 정량화 및 형태 학적 분석을 할 수 있습니다. </li></ol> Jagged1 코팅 된 표면과 노치 신호 전달의 5 연속 자극 <ol><li> 10 ㎍ / ml의 농도로 PBS에 염소 항 - 인간 IgG 항체의 Fc 일시 중단 (1 : 10 ㎎ / ㎖의 스톡 1000 희석). 1.3 μg의 / ㎠의 밀도로 배양 표면 항체 용액을 첨가하고 실온에서 1 시간 동안 배양한다. 24 웰 플레이트의 경우에, 웰 당 250 μL (2.5 μg의)를 추가한다. </li><li> 진공 흡인 우물과 1 ml의 PBS로 리필. 이 과정을 2 회 반복합니다. </li><li> 10 μg / ml의 농도에서 재조합 Jagged1 PBS-Fc 융합체 단백질을 일시. 1.3 μg의 / ㎠의 밀도에서, 항체 - 코팅 된 배양 표면 Jagged1-FC 용액을 첨가하고 실온에서 2 시간 동안 배양한다. 배양 표면 항체 ONL 도포Y는 컨트롤로서 사용될 수있다. </li><li> 진공 흡인 우물과 1 ml의 PBS로 리필. 이 과정을 2 회 반복합니다. 건조에서 그들을 방지하기 위해 시드 셀하기 직전까지 우물에서 PBS를 유지합니다. </li><li> 시드 2.6 × 10 4 파골 세포 전구체 / 코팅 표면 위에 cm 2. 노치 신호 전달은 연속적으로 적어도 3 일간 자극한다. </li></ol> Jagged1 코팅 비즈와 노치 신호 전달 6. 임시 자극 <ol><li> 1.5 ㎖의 최종 부피로 0.5 μg의 / cm 2 Jagged1-FC, 21 μL / cm 2 G 단백질 아가 로스 비드, 및 PBS : 적절한 크기의 튜브에 다음을 결합한다. 24 웰 플레이트의 1도에 대한 Jagged1 구슬의 적절한 수를 준비 1.5 ml의 1 μg의 Jagged1-FC, 40 ㎕의 단백질 G 아가로 오스 비즈와 PBS를 결합합니다. </li><li> 4에서 회전 튜브를 품어 2 시간 동안 C를 °. </li><li> 펠렛 비즈에 1 분 300 XG에 원심 분리기 튜브. 1.5 ml의 PBS에 재현 탁 구슬. 이 세척을 반복2 번 이상. </li><li> 130 μL / cm 2 배지에서 재현 탁 Jagged1 구슬과 배양 세포에 적용됩니다. </li><li> 원하는 기간 동안 CO 2를 37 ° C에서 구슬과 세포를 품어 5 %. PBS 또는 배지 여러 세척과 구슬을 제거합니다. </li><li> 노치 대상 유전자 전 사체 (10)의 측정을 통해 노치 신호 전달 활성화를 확인합니다. </li></ol>

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G., Kang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-derived+JAGGED1+promotes+osteolytic+bone+metastasis+of+breast+cancer+by+engaging+notch+signaling+in+bone+cells.">Tumor-derived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells.</a> Cancer Cell. 19 (2), 192-205 (2011).</li><li>Sekine, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+osteoclastogenesis+by+Notch2/Delta-like+1+and+Notch1/Jagged1+axes.">Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes.</a> Arthritis Res Ther. 14 (2), R45 (2012).</li><li>Ashley, J. W., Ahn, J., Hankenson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+Signaling+Promotes+Osteoclast+Maturation+and+Resorptive+Activity.">Notch Signaling Promotes Osteoclast Maturation and Resorptive Activity.</a> J Cell Biochem. 116 (11), 2598-2609 (2015).</li><li>Fukushima, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+association+of+Notch2+and+NF-kappaB+accelerates+RANKL-induced+osteoclastogenesis.">The association of Notch2 and NF-kappaB accelerates RANKL-induced osteoclastogenesis.</a> Mol Cell Biol. 28 (20), 6402-6412 (2008).</li><li>Canalis, E., Schilling, L., Yee, S. P., Lee, S. K., Zanotti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hajdu+Cheney+Mouse+Mutants+Exhibit+Osteopenia,+Increased+Osteoclastogenesis,+and+Bone+Resorption.">Hajdu Cheney Mouse Mutants Exhibit Osteopenia, Increased Osteoclastogenesis, and Bone Resorption.</a> J Biol Chem. 291 (4), 1538-1551 (2016).</li></ol>

The authors declare that they have no competing financial interests.

프로토콜 내에서 중요한 단계 문화와 파골 세포 전구체의 체외 분화는 파골 세포 및 골 재생과 뼈 질량의 보존을위한 치료 대상의 식별 분자 메커니즘의 조사를위한 유용한 플랫폼을 제공합니다. 마우스 파골 세포 전구체를 배양 할 때, 가장 중요한 요소는 나이브 상태의 전구체를 유지한다. 마크로파지 유사 세포로, 파골 세포 전구체는 박테리아 성분 및 염증성 사이토...

포유류 노치 신호 전달 경로는, 초파리 melanogaster의에서 동일한 경로에 상동이며, 네 횡단 노치 수용체 (Notch1-4) 및 가변 (JAG1 및 JAG2) 및 델타처럼 (DLL1, 3의 다섯 막 결합 리간드로 구성 및 4) 가족 (1). 수신 셀에 노치 수용체가 투과 셀이 리간드에 의해 결합 될 때 노치 신호 전달이 개시된다. 이 트랜스 활성화 중에 막 결합 노치 리간드는 막 결합 수용체의 노치 (3, 4)에 인장력을 생성한다. 리간드 결합의 인장력은 프레 함유 감마 - 세 크레타 제 복합체 (γ 세 크레타 제)에 의해 매개되는 세포 내 절단 이벤트 뒤에 효소 (TACE)에 변환 TNF- 알파에 의해 수용체의 세포 분열을 촉진 노치 수용체의 구조적 변화를 유도한다. γ 세 크레타 자료 노치 INT이 CBF -1- 쓰 (H) - lag 여기서 -1- (CSL), 배후 형상 (MAML)과 전사 활성 복합체를 형성하는 핵으로 전위 시키며, 세포 유형 특이 적 인자 발현을 구동 racellular 도메인 (NICD) 유전자 5를 대상으로. 시험관 내에서 노치 경로의 활성화 방법의 고유 한 필요성 노치 신호 전달 활성 결과의 기계적 요소. 가용성 노치 리간드는 수용체를 노치에 결합하지만, 동시에 경쟁적으로 세포 관련 노치 리간드의 결합을 억제하면서 NICD 해제에 필요한 힘을 스트레칭 생산하기 위해 실패 할 수 있습니다. 따라서, 배양 배지에 용해 노치 리간드 첨가 6,7 시그널링 정상 노치를 감쇄 할 수있다. 이들이 적절 리지드 기판 (5), (8, 9)에 고정되는 경우 다행히도 노치 리간드 NICD 방출을 유도 할 수있다,10. 리간드 피복 된 배양 세포를 기판 상에 시드 또는 세포 리간드 피복 된 비드를 적용 모두 노치 신호를 활성화하고, 그들 사이의 선택은 노치 자극 원하는 타이밍에 주로 의존 할 수있다. 기능 분석 또는 분화의 중간 동안 소망되는 바와 같이 즉각적인 임시 노치 신호의 활성화를 들어, 노치 리간드는 배양 된 세포에 적용 비드를 아가 로스에 바인딩 될 수 있고 언제든지 세척. 배양 기간의 시작 부분에서 더 지속 노치 시그널링, 조직 배양 플레이트를 세포 시딩 이전 리간드로 코팅 될 수있다. 이 프로토콜의 목적을 위해, 방법은 마우스 파골 세포 전구체를 사용하여 수행되지만, 여기에 기술 된 방법에 대한 방법과 유사 세포 타입 6, 11, 12, 13의 다양한 적용하고, </SUP&gt; 14. 파골 세포는 말단 골 조직의 재 흡수에 대한 책임 분화 조혈 세포 계보이며, 그들은 골 소실 (15)의 여러 질환에 연루되어있다. 따라서, 자신의 단핵구 / 대 식세포 계통의 전구 물질로부터 파골 세포와 그 기능이 파골 세포와 새로운 뼈 재생 치료의 개발의 더 나은 이해에 필수적인 제어하는 분자 메커니즘의 분화의 생체 외 연구한다. 지금 일반적 해당 노치 시그널링은 파골 세포의 분화와 기능에 긍정적 인 역할을 허용하는 동안, 모두 노치 신호 자극 및 파골 세포 전구체의 배양과 분화의 변화는 초기 상반된 결과 16, 17, 18, 19되었다. 가까이 방법의 차이 검사와 유전자의 사용모델은 크게 파골 노치 신호 전달의 역할을 명확히했지만, 표준화 노치 자극 배양 방법의 적용은 다른 세포 유형 20, 21, 22, 23 노치 신호의 향후 연구에서 이러한 논쟁을 방지 할 수있다. 노치 신호 전달을 자극위한 다양한 방법과 같이, 가장 좋은 방법은 실험 문제에 달려있다 배양 마우스 파골 세포 전구체의 분화를위한 여러 방법이 있으며. 여기서, 마우스 긴 뼈에서 플러시 골수 세포의 부착 및 비 부착 분수를 배양하는 우리의 선호하는 방법이 표시됩니다. 이 방법은 분화 된 다양한 방법에 적용 할 수있는 세포를 본질적으로 특별한 장비가 필요없고, 제조의 이점을 갖는다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Recombinant mouse M-CSF</td>
			<td>Biolegend</td>
			<td>576402</td>
			<td>Available from multiple suppliers, test activity before experiments</td>
		</tr>
		<tr>
			<td>Recombinant mouse RANKL</td>
			<td>Shenandoah Biotechnology</td>
			<td>200-04</td>
			<td>Available from multiple suppliers, test activity before experiments</td>
		</tr>
		<tr>
			<td>Recombinant human Jagged1-Fc</td>
			<td>R&#38;D Systems</td>
			<td>1277-JG-050</td>
			<td>Available from multiple suppliers, test activity before experiments</td>
		</tr>
		<tr>
			<td>Protein G agarose beads</td>
			<td>InvivoGen</td>
			<td>gel-agg-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-human IgG Fc</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-001-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium powder</td>
			<td>Sigma-Aldrich</td>
			<td>M0894</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase cell dissociation reagent</td>
			<td>ThermoFisher</td>
			<td>A1110501</td>
			<td>Used to lift osteoclast precursors</td>
		</tr>
		<tr>
			<td>Acid Phosphatase, Leukocyte (TRAP) Kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A-1KT</td>
			<td>Used to stain differentiated osteoclasts</td>
		</tr>
	</tbody>
</table>

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.

이 방법의 목적은 문화이며, 파골 세포 전구체에 노치 신호를 자극한다. 때 제대로 배양, 파골 세포 전구체는 부드러운 세포질 (그림 1A)와 주로 긴 스핀들 모양의 형태를 나타낸다. 주의가 파골 세포 전구체의 면역 활성화를 피하기 위해주의해야한다. 활성화되면, 전구체 확산과 거품 세포질 (그림 1B)로 평평하게됩니다. 이러한 &quot;기름에 튀긴 ?...

Watch this Scientific Journal Video about Stimulation of Notch Signaling in Mouse Osteoclast Precursors at JoVE.com

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

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.

마우스 파골 세포 전구체의 노치 신호 전달의 자극

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

stimulation-of-notch-signaling-in-mouse-osteoclast-precursors

Research

JoVE Journal

Developmental Biology

7.8K 조회수.  Perelman School of Medicine, University of Pennsylvania. 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.

비디오: Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

흉선의 특성 사용하여 마우스 배아에서 전구 세포를 정착<em&gt; 생체</em&gt;와<em&gt; 체외</em&gt; 분석 실험

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

연구

발생학

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

The canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue.

분리 및 성인 개 해마 신경 전구체의 확장

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

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.

약물 치료와<em&gt; 생체</em&gt;은 메다카 물고기 골다공증 모델에서 조골 파골 세포 상호 작용의 영상

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

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Mouse Secondary Palate Fusion의 라이브 이미징

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

마우스에서 초기 임신 특성에 대 한 3 차원 높은-주파수 초음파의 새로운 사용

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

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

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

RANKL 기반 Osteoclast 문화 분석 결과의 마우스 골 수 mTORC1 Osteoclast 형성에의 역할을 조사 하

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

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

절연, 문화, 및 골 수 기질 세포와 생쥐에서 Osteoclast 창시자의 차별화

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

트랜스와 초파리 노치의 바인딩을 평가를 집계 분석 셀-Ligands 및 Cis에 의해 그것의 억제-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

프로 거리 신호 억제 유도 Cardiomyocytes에 마우스 미 발달 섬유 아 세포의 프로그래밍 요소 중재 Potentiates

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

노드 및 스캐닝 전자 현미경 검사 법, 면역 형광 산 전체를 사용 하 여 Gastrulating 마우스 배아에서 Notochordal 접시를 시각화

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

마우스 파골 세포 전구체의 노치 신호 전달의 자극

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