Authors would like to thank Catherine Solioz for her skillful assistance.

1. BCM 준비 <ol><li> 가능한 한 신선한 지역 정육점에서 돼지 턱을 얻습니다. 회사 표면에 턱을 놓고 전체 두께 플랩이있는 부드러운 조직을 떠나거나 뼈에 부착 된 골막하지 않도록 특별한주의를 기울이고 놓습니다. 흐름 후드에서 작동 할 필요없이 깨끗한 환경에서 작업. </li><li> 전체 두께 플랩이 해제되면, 협측에서 뼈 칩을 수확 뼈 스크레이퍼를 사용합니다. 뼈 스크레이퍼 날카로운되어 있어야합니다. 단단히 뼈 스크레이퍼를 처리하고 긴 운동은 뼈를 수집로. 작은 그 1mm를 뼈 칩을 폐기하십시오. <ol><li> 기본 뼈 칩을 유지하기 위해, 1 %의 항생제와 그들을 밖으로 건조를 허용하지 않습니다 항진균제 보충 둘 베코의 수정 이글의 중간 (DMEM)와 10cm 직경의 플라스틱 접시에 직접 뼈 칩을 놓습니다. </li><li> 열처리의 영향을 평가하기 위해, 대상 뼈 칩은 80 ° C에서 30 분 동안 저온 살균 또는121 ℃에서 20 분 동안 오토 클레이브. </li><li> , 탈회의 영향을 평가 4 ℃에서 4-6 시간 동안 1 M HCL에 뼈 칩을 흔들 pH가 중성이 될 때까지 배양 배지로 반복 세척합니다. </li></ol></li><li> 새로운 플라스틱 접시에 1 % 항생제와 항진균제로 보충 된 10 ml의 신선한 DMEM 당 뼈 칩 5 g의 총을 놓습니다. </li><li> 24 시간 동안 37 ℃에서 가습 분위기에서 플라스틱 접시를 놓습니다. 그런 다음, 수확 BCM. 원심 분리기, 파편을 제거 멸균 (0.2 nm의) 필터, -80 ℃에서 냉동 분취 량을 유지하기 위해 10 분 동안 200 XG에 BCM. </li><li> 바로 사용하기 전에 BCM 주식을 해동과 동결 융해의 반복주기를 피하십시오. </li><li> 나타낸 실험에서, RT에서 1 시간 동안 BCM 또는 무 혈청 배지로 콜라겐 막을 담가. PBS로 적극적으로 막을 세척하고 96 웰 플레이트에 배치합니다. 젖은 막은 세포와 씨앗을 품고있다. </li><li> BCM의 제조 공정은도 1에 요약 하였다. </리&gt; </ol> 간엽 세포 기준 2. 생물학적 정량 <ol><li> / cm 2 30,000 세포의 농도로 12 웰 플레이트 (예를 들어, 골 세포, 치은 및 치주 인대 섬유 아세포) 종자 인간 간엽 세포. 세포를 DMEM으로 이루어지는 성장 배지, 10 % 소 태아 혈청과 항생제를 사용 시드. 세포는 플레이트의 O / N에 부착하자. </li><li> 문화 매체를 취소하고 37 ° C에서 미리 예열 PBS로 세포를 씻어. 와 20 % BCM없이 예열 무 혈청 배지를 첨가하여 세포를 자극한다. 24 시간 동안 37 ° C에서 가습 된 분위기에서 셀을 놓는다. </li><li> , 문화 매체를 폐기 미리 예열 PBS로 세포를 씻어하고 원하는 프로토콜에 따라 RNA를 추출. </li><li> 각 샘플에서 RNA의 동일한 금액을하기 위해 RNA의 농도를 조정합니다. cDNA를 준비하고, 표 1에 나타낸 프라이머를 사용하여 선택된 유전자를 분석 QRT-PCR을 수행한다. 참고 :이 모든 구성 요소의 희석 수 있습니다 : 배 SYBR 녹색, 20 배 프라이머 앞으로, 20 배 프라이머 역, 5 배 무균 DD 물, 5 배의 cDNA. QRT-PCR은 95 ° C 15 초, 40 사이클 및 60 ° C에서 1 분간 수행된다. </li><li> CT GAPDH와 Δ (ΔCT) ΔCT 자극 - - ΔCT 제어 ΔCT는 CT의 대상이되는 Δ (ΔCT) 방법을 사용하여 하우스 키핑 유전자 GAPDH에 정상화가 상대적 발현 수준을 계산합니다. </li><li> 이 품질 관리 후에, 중간 엽 세포, 조혈 세포 또는 내피 세포를 포함하여 모든 유형의 세포를 자극하는 배양액을 추가로 BCM. </li></ol>

<ol>
	<li>Buser, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+Stability+of+Early+Implant+Placement+with+Contour+Augmentation.">Long-term Stability of Early Implant Placement with Contour Augmentation.</a> Journal of dental research. , (2013).</li><li>Chiapasco, M., Casentini, P., Bone Zaniboni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=augmentation+procedures+in+implant+dentistry.">augmentation procedures in implant dentistry.</a> The International journal of oral &amp; maxillofacial implants. 24 Suppl. , 237-259 (2009).</li><li>Giannoudis, P. V., Dinopoulos, H., Bone Tsiridis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=substitutes:+an+update.+Injury.">substitutes: an update. Injury.</a> 36, Suppl 3. S20-S27. , (2005).</li><li>Jensen, S. S., Broggini, N., Hjorting-Hansen, E., Schenk, R., Bone Buser, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=healing+and+graft+resorption+of+autograft,+anorganic+bovine+bone+and+beta-tricalcium+phosphate.+A+histologic+and+histomorphometric+study+in+the+mandibles+of+minipigs.">healing and graft resorption of autograft, anorganic bovine bone and beta-tricalcium phosphate. A histologic and histomorphometric study in the mandibles of minipigs.</a> Clinical oral implants research. 17, 237-243 (2006).</li><li>Jensen, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+novel+biphasic+calcium+phosphate+in+standardized+bone+defects:+a+histologic+and+histomorphometric+study+in+the+mandibles+of+minipigs.">Evaluation of a novel biphasic calcium phosphate in standardized bone defects: a histologic and histomorphometric study in the mandibles of minipigs.</a> Clinical oral implants research. 18, 752-760 (2007).</li><li>Grabowski, G., Bone Cornett, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=graft+and+bone+graft+substitutes+in+spine+surgery:+current+concepts+and+controversies.">graft and bone graft substitutes in spine surgery: current concepts and controversies.</a> The Journal of the American Academy of Orthopaedic Surgeons. 21, 51-60 (2013).</li><li>Khan, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+bone+grafting.">The biology of bone grafting.</a> The Journal of the American Academy of Orthopaedic Surgeons. 13, 77-86 (2005).</li><li>Bohr, H., Ravn, H. O., Werner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+osteogenic+effect+of+bone+transplants+in+rabbits.+The+Journal+of+bone+and+joint+surgery.">The osteogenic effect of bone transplants in rabbits. The Journal of bone and joint surgery.</a> British. 50, 866-873 (1968).</li><li>Bone Urist, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=formation+by+autoinduction.">formation by autoinduction.</a> Science. 150, 893-899 (1965).</li><li>Caball&#233;-Serrano, J. D., Buser, D., Gruber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+porcine+bone+conditioned+medium.">Proteomic analysis of porcine bone conditioned medium.</a> The International journal of oral &amp; maxillofacial implants. , (2014).</li><li>Peng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone-Conditioned+Medium+Inhibits+Osteogenic+and+Adipogenic+Differentiation+of+Mesenchymal+Cells+In+Vitro.">Bone-Conditioned Medium Inhibits Osteogenic and Adipogenic Differentiation of Mesenchymal Cells In Vitro.</a> Clinical implant dentistry and related research. , (2014).</li><li>Brolese, E., Buser, D., Kuchler, U., Schaller, B., Gruber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+bone+chips+release+of+sclerostin+and+FGF-23+into+the+culture+medium:+an+in+vitro+pilot+study.">Human bone chips release of sclerostin and FGF-23 into the culture medium: an in vitro pilot study.</a> Clinical oral implants research. , (2014).</li><li>Caball&#233;-Serrano, J., Bosshardt, D. D., Gargallo-Albiol, J., Buser, D., Bone Gruber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=conditioned+medium+enhances+osteoclastogenesis+in+murine+bone+marrow+cultures.+Clin+Oral+Impl+Res;+in.">conditioned medium enhances osteoclastogenesis in murine bone marrow cultures. Clin Oral Impl Res; in.</a> Int J Oral Maxillofac Surg. , (2015).</li><li>Zimmermann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone-conditioned+medium+changes+gene+expression+in+bone-derived+fibroblasts).">Bone-conditioned medium changes gene expression in bone-derived fibroblasts).</a> IJOMI accepted. , (2014).</li><li>Fulzele, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelopoiesis+is+regulated+by+osteocytes+through+Gsalpha-dependent+signaling.">Myelopoiesis is regulated by osteocytes through Gsalpha-dependent signaling.</a> Blood. 121, 930-939 (2013).</li><li>Becerra, J., Andrades, J. A., Ertl, D. C., Sorgente, N., Nimni, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demineralized+bone+matrix+mediates+differentiation+of+bone+marrow+stromal+cells+in+vitro:+effect+of+age+of+cell+donor.">Demineralized bone matrix mediates differentiation of bone marrow stromal cells in vitro: effect of age of cell donor.</a> Journal of. 11, 1703-1714 (1996).</li><li>Kupcova Skalnikova, ., H, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+techniques+for+characterisation+of+mesenchymal+stem+cell+secretome.">Proteomic techniques for characterisation of mesenchymal stem cell secretome.</a> Biochimie. , (2013).</li><li>Romanello, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblastic+cell+secretome:+A+novel+role+for+progranulin+during+risedronate+treatment.">Osteoblastic cell secretome: A novel role for progranulin during risedronate treatment.</a> , (2013).</li><li>Schuldt Filho, ., G, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+medium+of+demineralized+bone+matrix+activates+TGF-&#946;+signaling+pathways+in+mesenchymal+cells+in+vitro.">Conditioned medium of demineralized bone matrix activates TGF-&#946; signaling pathways in mesenchymal cells in vitro.</a> J Cranio Maxill Surg. , (2015).</li><li>Buser, D., Chen, S. T., Weber, H. P., Belser, U. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+implant+placement+following+single-tooth+extraction+in+the+esthetic+zone:+biologic+rationale+and+surgical+procedures).">Early implant placement following single-tooth extraction in the esthetic zone: biologic rationale and surgical procedures).</a> The International journal of periodontics &amp; restorative dentistry. 28, 441-451 (2008).</li><li>Stoecklin-Wasmer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absorbable+collagen+membranes+for+periodontal+regeneration:+a+systematic+review.">Absorbable collagen membranes for periodontal regeneration: a systematic review.</a> Journal of dental research. 92, 773-781 (2013).</li></ol>

저자가 공개하는 게 없다. 조르디 Caballé - 세라노는 치과 연구 및 교육, 바젤, 스위스의 재단에서 장학금을 받았다.

뼈 에어컨 매체는 골 재생의 초기 단계 동안 뼈 이식의 공개 활동을 반영한다. 여기에 설명 된 프로토콜이 골 재생에 관여하는 세포의 다른 유형의 반응을 연구하기 위해 적응 될 수있다. 또한, 프로토콜 처리 된 뼈 또는 뼈 충전제로부터의 조건화 배지를 제조 할 수있다. 다양한 네이티브 및 처리 뼈에서 방출 요인 : 방법을 수행하고 간단한 개념에 의존하기 쉽다. 방법 BCM을 이해하는 것은 중간 ?...

자가 뼈 널리 기형의 결과, resective 수술, 재건 외상 수술, 이전 임플란트 식립 1, 2에로 발생한 결함을 연결하는 데 사용됩니다. 뼈 이식은 이식 통합의 과정을 지원하는 방법의 생물학적 원리를 이해하는 것은자가 이식이 재건 수술의 황금 표준으로 간주하는 이유를 이해하기 만 키가 아닌, 그것은 또한 뼈 대체 (3)의 개선 된 디자인에 생체 공학입니다. 그러나 이식 통합은 빠른자가 뼈와 뼈가 4,5 대체 비교된다. 따라서, 골 재생을 지원하기 때문에 효과적인 자가골을 분자 세포 메커니즘을 나타 내기 위해 필수적이다. 통합 프로세스의 6,7을 지원하는 것으로 간주되는자가 이식의 세 교과서의 특성이 있습니다. 첫째,자가 뼈가 성장하는 새로 형성된 뼈에 대한 지침을 제공, osteoconductive입니다결함에. 둘째,자가 뼈는 조골 세포 (8)로 분화 할 수 중간 엽 세포를 포함 즉, 골 형성이다. 매트릭스 내에 안치 뼈 형태 형성 단백질 유사 성장 인자 연골 또는 골 형성 intramembranous (9)의 처리를 개시 할 수있다 셋째, 자가골은 골 유도된다. 또 다른 측면이있다 : 갓 준비 뼈 칩은 &quot;뼈 에어컨 매체&quot;10 ~ 15과 체외 관찰을 기반으로 분비 기능을 유지. 또한 myelopoiesis의 영향은 16를 언급한다. &quot;탈회 골 기질 - 조정 배지&quot;A와 유사한 용어는 이미 탈회 (17)에 의해 처리 된 경우에도, 1996 년 낸 뼈의 분비 기능의 전체적인 개념을 지원 하였다. 우리의 목적을 위해, BCM은 10,11 하악골 신선한 돼지로부터 제조 될 수있다. BCM의 프로테옴 분석은 성장의 사실을 포함하여, 복잡한 구성을 밝혀ORS과 세포 외 기질 (10)의 성분은, 또한 전체의 뼈 18,19 테아의 기존 기술을 연장. 따라서, BCM은 시험관 내에서 뼈 이식의 다양한 변형의 공개 활동을 반영해야한다. 중간 엽 세포는, 예를 들어 뼈 칩 또는 구두 부드러운 조직에서 분리 된 사람들을 위해, BCM 수 있나요? 체외 노출되었을 때 어떻게됩니까, BCM은 골 형성과 지방 세포 분화를 감소시키고, IL11 식 (11)의 강한 증가를 불러 일으킨다. 게놈 넓은 마이크로 어레이는 차등 BCM에 대한 응답으로 중간 엽 세포에서 발현되는 많은 유전자를 한 것으로 밝혀졌습니다. 이들 유전자 중 adrenomedullin (ADM)이며, IL11, IL33, NADPH 산화 효소 4 (NOX4), 프로테오글리칸 4 (PRG4, 또는 lubricin) 및 3 (PTX3) 15 pentraxin. 멸균 뼈 칩으로부터 얻어진 BCM은 각각 14 유전자의 발현을 변경하지 못한 경우. 저온 살균을 시행하고 동결 뼈 칩에서 BCM은 할 수 있었다유전자 발현 (14)을 변경합니다. 탈회 골 기질 (DBM-CM)의 또한 에어컨 매체는 TGF-β 조절 유전자 (20)의 표현을 변경합니다. 흥미롭게도, 주위의 부드러운 조직 뼈 (21, 22)로부터 칩을 보호하는 데 콜라겐 배리어 막은, 유전자 발현의 변화 (23)에 대한 책임 BCM의 그 부분을 흡착. BCM 연구는 몇 가지 이름을, 같은 뼈 재 흡수 파골 세포와 내피 세포로 골 재생에 관여하는 다른 세포 유형으로 확장 할 수 있습니다. 전반적으로, 축적 체외 데이터는 임상 연구의 설계를위한 과학적 근거를 제공한다. 본 프로토콜은 두 가지입니다 : 첫째, BCM을 준비하는 방법을 보여줍니다. 둘째, 체외에서 중간 엽 세포를 기반으로 생물학적 활성을 테스트하는 방법을 보여줍니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pig Mandibles</td>
			<td>Local bucher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bone Scraper</td>
			<td>Hu-Friedy</td>
			<td>PPBUSE2/36</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotics &#38; Antimicotics</td>
			<td>All life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Membranes (Bio-Gide)</td>
			<td>Geistlich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Invitrogen Corporation</td>
			<td align="right">16030074</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen Corporation</td>
			<td>21885-025</td>
			<td></td>
		</tr>
		<tr>
			<td> 
			High Pure RNA Isolation Kit</td>
			<td>Roche</td>
			<td align="right">11828665001</td>
			<td></td>
		</tr>
		<tr>
			<td>Transcriptor First Strand cDNA Synthesis Kit</td>
			<td>Roche</td>
			<td align="right">4379012001</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>Microsynth</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green (for Q-RT-PCR)</td>
			<td>Roche</td>
			<td align="right">4673484001</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Roche</td>
			<td align="right">11666789001</td>
			<td></td>
		</tr>
	</tbody>
</table>

bone conditioned medium: preparation and bioassay

자가 뼈 이식 널리 구강 악안면 외과, 정형 외과 및 외상에 사용됩니다. 자가 뼈 이식은 누락 된 뼈를 대체하지, 그들은 또한 골 재생의 복잡한 과정을 지원합니다. 자가 이식이 유리한 동작은 세 가지 특징에 기인한다 : osteoconductivity, osteogenicity 및 osteoinductivity을. 그러나, 다른 측면이있다 : 뼈 골 재생에 관련된 중간 엽 세포를 대상으로 할 수 성장 인자를 포함하여 분자의 무수 해제 접목. 골 이식의 분비 속성은 뼈 에어컨 매체 (BCM)를 사용하여 시험 관내에서 공부하실 수 있습니다. 여기에서 우리는 열 처리 또는 탈회를 시행 기본 돼지 피질 뼈에서 뼈 에어컨 매체를 준비하는 방법에 대한 프로토콜 및 뼈를 제시한다. 세포는 직접 BCM에 노출 또는 콜라겐 멤브레인, 이전 BCM 적신 같은 생체 재료,에 접종 할 수있다. 우리는 체외 bioassa에 대한 예를들TGF-β 조절 유전자의 발현에 중간 엽 세포와 YS. 제시된 프로토콜은 추가 골 재생시 뼈 이식의 분비 효과를 밝혀 및 재건 수술의 폭 넓은 분야의 중개 연구에 대한 경로를 엽니 다 장려해야한다.

뼈 조정 배지를 신선한 돼지 뼈 칩으로부터 제조된다. 공정의 일반 개요 BCM을 준비하고 BCM 생체 물질과 조합을 사용은도 1 및도 2에 각각 도시되어있다. BCM 준비 동안, 최종 BCM의 품질에 영향을 미칠 수있는 짧은 움직임 또는 매우 작은 뼈 칩만큼 움직임 큰 뼈 칩을 얻는 것이 중요하다. ADM, PTX3, IL11, IL33, NOX4 및 PRG4 (도 3) : BCM의 품질 BCM 타겟 유전자의 유전자 발현을 분석하?...

Watch this Scientific Journal Video about Bone Conditioned Medium: Preparation and Bioassay at JoVE.com

Bone Conditioned Medium: Preparation and Bioassay

우리는 뼈 에어컨 매체 (BCM)을 준비하고 시험 관내에서 활동을 테스트하는 방법을 여기에 대해 설명합니다.

뼈 조정 배지 : 준비 및 생물 검정

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing ...

bone-conditioned-medium-preparation-and-bioassay

Research

JoVE Journal

Biology

11.0K 조회수.  School of Dental Medicine, University of Bern.  우리는 뼈 에어컨 매체 (BCM)을 준비하고 시험 관내에서 활동을 테스트하는 방법을 여기에 대해 설명합니다. 

비디오: Bone Conditioned Medium: Preparation and Bioassay

Maintaining Wolbachia in Cell-free Medium

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ascertain the viability of the purified Wolbachia and (3) maintain the now extracellular Wolbachia in cell-free medium.  Purified Wolbachia remain alive in the extracellular phase but do not replicate until re-inoculated into eukaryotic cells.  Extracellular Wolbachia purified in this manner will remain viable for at least a week at room temperature, and possibly longer.  Purified Wolbachia are suitable for micro-injection, DNA extraction and other applications. 


This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated.

셀없는 매체 Wolbachia을 유지

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ...

연구

생물학

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images1 and allows for techniques such as fluorochrome assessment and histomorphometry2. Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. 
Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.

Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.

조직학, 조직 형태 측정 및 분석을위한 Fluorochrome Undecalcified 본 준비

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Extracellular 소포 세포 조절 미디어에서의 Cytometric 분석 흐름

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

인간 골관절염 무릎 관절에서 조직 수집 및 RNA 추출

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

루트 엑수데이트에서 뿌리 균 화학 물질의 신속 한 식별에 대 한 향상 된 화학 분석

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with&#160;the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along&#160;a range of insecticide concentrations.
This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

모기 및 초파리에 대한 살충제 독성을 정량화하기위한 국소 응용 생물 검정

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. ...

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is limited by cost, throughput, and the caveats inherent to isotopic labeling of the food Fe. Thus, there exists a critical need for an approach that is high-throughput and cost-effective. The Caco-2 cell bioassay was developed to satisfy this need. The Caco-2 cell bioassay for Fe bioavailability utilizes simulated gastric and intestinal digestion coupled with culture of a human intestinal epithelial cell line known as Caco-2. In Caco-2 cells, Fe uptake stimulates the intracellular formation of ferritin, an Fe storage protein easily measured by enzyme-linked immunosorbent assay (ELISA). Ferritin forms in proportion to Fe uptake; thus, by measuring Caco-2 cell ferritin production, one can assess intestinal Fe uptake from simulated food digests into the enterocyte. 
Via this approach, the model replicates the key initial step that determines food Fe bioavailability. Since its inception in 1998, this model approach has been rigorously compared to factors known to influence human Fe bioavailability. Moreover, it has been applied in parallel studies, with three human efficacy studies evaluating Fe biofortified crops. In all cases, the bioassay correctly predicted the relative amounts of Fe bioavailability from the factors, crops, and overall diet. This paper provides detailed methods on Caco-2 cell culture coupled with the in vitro digestion process and cell ferritin ELISA necessary to conduct the Caco-2 cell bioassay for Fe bioavailability.

The Caco-2 cell bioassay for iron (Fe) bioavailability represents a cost-effective and versatile approach to assess Fe bioavailability from foods, food products, supplements, meals, and even diet regimens. Thoroughly validated to human studies, it represents the state of the art for studies of Fe bioavailability.

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is ...

내연골 골화술을 통한 뼈 재생을 위한 인공 연골 임플란트

Integrated Bone Formation Through In Vivo Endochondral Ossification Using Mesenchymal Stem Cells

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it does not have a mechanism to induce angiogenesis. Implanting artificial cartilage tissue fabricated from MSCs induces angiogenesis and bone formation in vivo via endochondral ossification (ECO). Therefore, this ECO-mediated approach may be a promising bone regeneration therapy in the future. An important aspect of the clinical application of this ECO-mediated approach is establishing a protocol for preparing enough cartilage to be implanted to repair the bone defect. It is especially not practical to design a single mass of grafted cartilage of a size that conforms to the shape of the actual bone defect. Therefore, the cartilage to be transplanted must have the property of forming bone integrally when multiple pieces are implanted. Hydrogels may be an attractive tool for scaling up tissue-engineered grafts for endochondral ossification to meet clinical requirements. Although many naturally derived hydrogels support MSC cartilage formation in vitro and ECO in vivo, the optimal scaffold material to meet the needs of clinical applications has yet to be determined. Hyaluronic acid (HA) is a crucial component of the cartilage extracellular matrix and is a biodegradable and biocompatible polysaccharide. Here, we show that HA hydrogels have excellent properties to support in vitro differentiation of MSC-based cartilage tissue and promote endochondral bone formation in vivo.

저자 스포트라이트: Vascularized Artificial Cartilage Integration을 통한 뼈 재생 향상 

Bone therapy via endochondral ossification by implanting artificial cartilage tissue produced from mesenchymal stem cells has the potential to circumvent the drawbacks of conventional therapies. Hyaluronic acid hydrogels are effective in scaling up uniformly differentiated cartilage grafts as well as creating integrated bone with vascularization between fused grafts in vivo.

중간엽 줄기세포를 이용한 In Vivo Endochondral Ossification Through Integrated Bone Formation

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it ...