Name: simple detection of primary cilia by immunofluorescence
Uploaded: 2020-05-15T14:00:00
Description: Watch this Scientific Journal Video about Simple Detection of Primary Cilia by Immunofluorescence at JoVE.com

이 작품은 체코 국방부에 의해 지원되었다 - 장기 조직 개발 계획 군사 보건 과학 학부의 대량 살상 무기의 의료 측면, 국방부; 교육부, 청소년 및 스포츠, 체코 (특정 연구 프로젝트 번호: SV / FVZ201703) 및 PROGRES Q40/06. 또한 다니엘 디아즈에게 영어 개정에 대한 그의 친절한 도움에 감사드립니다.

1. 문화 매체, 솔루션 및 요리 준비
<ol><li>커버립(22 x 22mm)을 자동 클래브합니다. 6 개의 잘 접시를 준비하십시오. 태아 소 혈청 (FBS) 및 항생제 페니실린 / 연쇄 절제술을 하고 문체 배지를 실온 (RT)으로 따뜻하게합니다. 트립신-EDTA 사용 (0.25%) 및 1x PBS (칼슘과 마그네슘을 곁들인 인산염 완충식식염)이 세포를 통과한다.</li>
	<li>dH2O (800 mg의 PFA 에서 dH2O)로 신선한 4 % 파라 포름알데히?...

<ol>
	<li>Alieva, I. B., Vorobjev, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertebrate+primary+cilia:+a+sensory+part+of+centrosomal+complex+in+tissue+cells,+but+a+"sleeping+beauty"+in+cultured+cells.">Vertebrate primary cilia: a sensory part of centrosomal complex in tissue cells, but a "sleeping beauty" in cultured cells.</a> Cell Biology International. 28 (2), 139-150 (2004).</li><li>Sloboda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Primary cilia. , (2009).</li><li>Sharma, N., Kosan, Z. A., Stallworth, J. E., Berbari, N. F., Yoder, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+levels+of+cytosolic+tubulin+regulate+ciliary+length+control.">Soluble levels of cytosolic tubulin regulate ciliary length control.</a> Molecular Biology of the Cell. 22 (6), 806-816 (2011).</li><li>Filipov&#225;, A., 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=Ionizing+radiation+increases+primary+cilia+incidence+and+induces+multiciliation+in+C2C12+myoblasts.">Ionizing radiation increases primary cilia incidence and induces multiciliation in C2C12 myoblasts.</a> Cell Biology International. 39 (8), 943-953 (2015).</li><li>Filipov&#225;, A., 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+toxic+effect+of+cytostatics+on+primary+cilia+frequency+and+multiciliation.">The toxic effect of cytostatics on primary cilia frequency and multiciliation.</a> Journal of Cellular and Molecular Medicine. 23 (8), 5728-5736 (2019).</li><li>Gadadhar, S., 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=Tubulin+glycylation+controls+primary+cilia+length.">Tubulin glycylation controls primary cilia length.</a> The Journal of Cell Biology. 216 (9), 2701-2713 (2017).</li><li>Shamloo, K., 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=Chronic+Hypobaric+Hypoxia+Modulates+Primary+Cilia+Differently+in+Adult+and+Fetal+Ovine+Kidneys.">Chronic Hypobaric Hypoxia Modulates Primary Cilia Differently in Adult and Fetal Ovine Kidneys.</a> Frontiers in Physiology. 8, 677 (2017).</li><li>Malone, A. M. D., 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=Primary+cilia+mediate+mechanosensing+in+bone+cells+by+a+calcium-independent+mechanism.">Primary cilia mediate mechanosensing in bone cells by a calcium-independent mechanism.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (33), 13325-13330 (2007).</li><li>Luesma, M. J., 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=Enteric+neurons+show+a+primary+cilium.">Enteric neurons show a primary cilium.</a> Journal of Cellular and Molecular Medicine. 17 (1), 147-153 (2013).</li><li>Pugacheva, E. N., Jablonski, S. A., Hartman, T. R., Henske, E. P., Golemis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEF1-dependent+Aurora+A+activation+induces+disassembly+of+the+primary+cilium.">HEF1-dependent Aurora A activation induces disassembly of the primary cilium.</a> Cell. 129 (7), 1351-1363 (2007).</li><li>Li, A., 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=Ciliary+transition+zone+activation+of+phosphorylated+Tctex-1+controls+ciliary+resorption,+S-phase+entry+and+fate+of+neural+progenitors.">Ciliary transition zone activation of phosphorylated Tctex-1 controls ciliary resorption, S-phase entry and fate of neural progenitors.</a> Nature Cell Biology. 13 (4), 402-411 (2011).</li><li>Spalluto, C., Wilson, D. I., Hearn, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+reciliation+of+RPE1+cells+in+late+G1+phase,+and+ciliary+localisation+of+cyclin+B1.">Evidence for reciliation of RPE1 cells in late G1 phase, and ciliary localisation of cyclin B1.</a> FEBS Open Bio. 3, 334-340 (2013).</li><li>Malicki, J. J., Johnson, 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=The+Cilium:+Cellular+Antenna+and+Central+Processing+Unit.">The Cilium: Cellular Antenna and Central Processing Unit.</a> Trends in Cell Biology. 27 (2), 126-140 (2017).</li><li>Morleo, M., Franco, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Autophagy-Cilia+Axis:+An+Intricate+Relationship.">The Autophagy-Cilia Axis: An Intricate Relationship.</a> Cells. 8 (8), 905 (2019).</li><li>Ott, C., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+live+primary+cilia+dynamics+using+fluorescence+microscopy.">Visualization of live primary cilia dynamics using fluorescence microscopy.</a> Current Protocols in Cell Biology. , (2012).</li><li>Sun, S., Fisher, R. L., Bowser, S. S., Pentecost, B. T., Sui, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+architecture+of+epithelial+primary+cilia.">Three-dimensional architecture of epithelial primary cilia.</a> Proceedings of the National Academy of Sciences. 116 (19), 9370-9379 (2019).</li><li>Lauring, M. 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=New+software+for+automated+cilia+detection+in+cells+(ACDC).">New software for automated cilia detection in cells (ACDC).</a> Cilia. 8 (1), 1 (2019).</li><li>Conroy, P. 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=C-NAP1+and+rootletin+restrain+DNA+damage-induced+centriole+splitting+and+facilitate+ciliogenesis.">C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis.</a> Cell Cycle. 11 (20), 3769-3778 (2012).</li><li>Kim, J. 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=Genome-wide+screen+identifies+novel+machineries+required+for+both+ciliogenesis+and+cell+cycle+arrest+upon+serum+starvation.">Genome-wide screen identifies novel machineries required for both ciliogenesis and cell cycle arrest upon serum starvation.</a> Biochimica et Biophysica Acta. 1863 (6), 1307-1318 (2016).</li><li>Yuan, K., 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=Primary+cilia+are+decreased+in+breast+cancer:+analysis+of+a+collection+of+human+breast+cancer+cell+lines+and+tissues.">Primary cilia are decreased in breast cancer: analysis of a collection of human breast cancer cell lines and tissues.</a> The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society. 58 (10), 857-870 (2010).</li><li>Smith, Q., 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+HDAC6+Activity+Modulates+Ciliogenesis+and+Subsequent+Mechanosensing+of+Endothelial+Cells+Derived+from+Pluripotent+Stem+Cells.">Differential HDAC6 Activity Modulates Ciliogenesis and Subsequent Mechanosensing of Endothelial Cells Derived from Pluripotent Stem Cells.</a> Cell Reports. 24 (4), 895-908 (2018).</li><li>Mirvis, M., Siemers, K. A., Nelson, W. J., Stearns, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cilium+loss+in+mammalian+cells+occurs+predominantly+by+whole-cilium+shedding.">Primary cilium loss in mammalian cells occurs predominantly by whole-cilium shedding.</a> PLOS Biology. 17 (7), 3000381 (2019).</li><li>Hua, K., Ferland, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+methods+can+differentially+affect+ciliary+protein+immunolabeling.">Fixation methods can differentially affect ciliary protein immunolabeling.</a> Cilia. 6 (1), 5 (2017).</li><li>Lim, Y. C., McGlashan, S. R., Cooling, M. T., Long, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+detection+of+primary+cilia+in+endothelial+cell+models.">Culture and detection of primary cilia in endothelial cell models.</a> Cilia. 4, 11 (2015).</li><li>DiDonato, D., Brasaemle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+methods+for+the+study+of+lipid+droplets+by+immunofluorescence+microscopy.">Fixation methods for the study of lipid droplets by immunofluorescence microscopy.</a> The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society. 51 (6), 773-780 (2003).</li></ol>

몇몇 저자는 1 차적인 섬모의 검출을 위한 다양한 방법을 기술했습니다, 때때로6또한 그들의 검출6,20,,21,,22에영향을 미칠 수 있는 각종 고정 방법을 기술합니다. 에 관계없이 탐지를 위한 완전하고 간단한 프로토콜을 찾기가 어렵습니다. 이러한 방법의 준비 가용성은 의심 할 여지없이 1 차 섬모 조사?...

1 차 적인 섬모는 세포의 어머니 중심과 관련 되 었던 감각, 독방, 막 바인딩, 비 momotile 구조. 1 차 섬모는 적혈구, 백혈구1,및 간세포2를제외한 대부분의 척추 동물 세포에서 발견된다. 1차 섬모는 마이크로튜부에 의해 구성된 길쭉한 축소메로 형성되며, 그 주요 성분은 α-tubulin이다. 축 사 메는 기저체에서 성장, 이는 γ-tubulin에서 구조화. 기본 섬모의 길이는 2-10 μm 사이다릅니다. 그러나, 그 치수는 글리시션, 기아, 저산소증, 세포독성 스트레스, 또는 이온화 방사선3,,4,,5,,6,,7에노출 된 후 변경 될 수 있습니다. 일반적으로, 세포는 세포 증식 및 분화8,,9에중요한 형태 발생 및 세포 신호 경로에 관여하는 단 하나의 1 차실을 갖는다.
1차 섬모는 세포 주기 진행 중에, 특히 G0/G1 단계 동안 동적으로 조절되며, HDAC6(히스톤 deacetylase 6)10에의해 매개되는 튜룰린 탈염과 관련된 과정에서 미토시스를 입력하기 전에 재조판된다. 1차 섬모 재흡수의 정확한 순간은 오로라 A, Plk1, TcTex-1 11,1112,13과같은 이 과정에 직접 관여하는 유전자의 세포 유형 및 발현에 따라 달라집니다.,, 세포 유형에 따라, 1 차 적인 섬모는 수용체의 다른 모형을 표현합니다, 이온 채널, 및 액티브 신호 통로. 이들은 증식및 생존에 영향을 미치는 가장 중요한 신호 수용체를 포함, EGFR, PDGFR, 및 FGFR. 또한 고슴도치, 노치 및 Wnt. 이러한 수용체 및 신호 경로 덕분에 하나 이상의 장기의 기능에 영향을 줄 수 있는 신호 경로 중 일부가 포함되어 있으며, 1차 섬모는 또한 화학 감각 기능을 수행합니다. 이 기능을 통해 1 차 적인 섬모 는 노치에 대 한 특정 리간드를 감지 할 수 있습니다., 호르몬, 그리고 세로토닌 또는 somatostatin 같은 생물학적 활성 물질. 다른 길이의 기본 섬모에 의해 전시 된 다른 특정 기능은 온도, 중력 및 진동(14)의변화에 대한 반응을 포함한다.
1차 섬모는 라이브 시각화, 전송 전자 현미경, 3D 이미징, 또는 1차,섬모5,15,,5,16,17의자동 검출을 위한 소프트웨어에 의해 다양한 방법을 통해 시각화될 수 있다. 그러나, 이러한 방법은 고도로 전문화 하 고 지속적인 연구 연구의 모든 단계에서 기본, 신속 하 고 쉬운 방법을 필요. 기재된 배양된 세포에서 1차 섬모를 검출하기 위한 쉽고 유용한 방법이기 다.

<table border="0" cellpadding="0" cellspacing="0" style="width:1301px;" width="1301">
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		<tr height="21">
			<td height="21" style="height:21px;width:384px;">6-well plate</td>
			<td style="width:217px;">TPP</td>
			<td style="width:119px;">92406</td>
			<td style="width:581px;">Dimensions 128x86x22 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Alexa Fluor488</td>
			<td style="width:217px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">111-546-047</td>
			<td>AffiniPure F(ab&#39;)&#8322; Fragment Goat Anti-Rabbit IgG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Anti-Tubulin &#947;</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">T5192</td>
			<td>Polyclonal Rabbit anti-Mouse IgG2a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">C2C12</td>
			<td style="width:217px;">ATCC</td>
			<td style="width:119px;">CRL-1772</td>
			<td>Myoblast (mouse)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Cy3</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">C2181</td>
			<td>Anti-Mouse IgG (whole molecule) F(ab&#8242;)2 fragment&#8211;Cy3 antibody produced in sheep</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Dapi (4&#8242;,6-Diamidino-2-phenylindole dihydrochloride)</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Dulbecco&#180;s Modified Eagle&#180;s medium</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">11960044</td>
			<td>High glucose, No glutamine, Gibco</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:384px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8662</td>
			<td>With MgCl2 and CaCl2, Sterile-filtered, Suitable for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Fetal Bovine Serum</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">16000044</td>
			<td>Sterile-Filtered, Gibco</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">L-Glutamine</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">MEF</td>
			<td style="width:217px;">ATCC</td>
			<td style="width:119px;">SCRC-1039</td>
			<td>Mouse embryonic fibroblast</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Monoclonal Anti-Acetylated Tubulin</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">T7451</td>
			<td>Monoclonal Anti-Acetylated Tubulin antibody produced in mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">NHLF</td>
			<td style="width:217px;">Lonza</td>
			<td style="width:119px;">CC-2512</td>
			<td>Primary lung fibroblasts (human)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Normal Goat Serum</td>
			<td style="width:217px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">005-000-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Paraformaldehyde</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">158127-500G</td>
			<td>Powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Penicillin-Streptomycin</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">P0781</td>
			<td>10,000 units penicillin and 10 mg streptomycin per mL in 0.9% NaCl, Sterile-Filtered</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">ProLong Diamond Antifade Mountant</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">P36961</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:384px;">Skin fibroblasts</td>
			<td style="width:217px;">Kindly gifted from Charles University, Faculty of Medicine in Hradec Kr&#225;lov&#233;.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Square Cover Slips</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">22X22-1.5</td>
			<td>Borosilicate glass, 22x22mm, Square</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Triton X-100</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">11332481001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">25200072</td>
			<td>Sterile-Filtered, Gibco</td>
		</tr>
	</tbody>
</table>

simple detection of primary cilia by immunofluorescence

1 차 적인 섬모는 세포 주기 진행 도중 동적으로 통제됩니다, 특히 세포 주기의 G0/G1 단계 도중, 미토시스의 앞에 resorbed되고. 1 차 섬모는 전송 전자 현미경 검사법, 3D 이미징, 또는 1 차 섬모의 자동 검출을 위한 소프트웨어를 사용하는 것을 포함하여 고도로 정교한 방법으로 시각화될 수 있습니다. 그러나, 이러한 방법을 수행하기 위해서는 1차 섬모의 면역형성 염색이 필요하다. 이 간행물은 아세틸화 알파 튜룰린 (axoneme)과 감마 튜룰린 (기저 몸)을 염색하여 체외에서 1 차 섬모를 쉽게 검출하기위한 프로토콜을 설명합니다. 이 면역 형광 염색 프로토콜은 비교적 간단하고 고품질의 이미지를 초래합니다. 본 프로토콜은 1차 섬모를 발현하는 4개의 세포주(C2C12, MEF, NHLF 및 피부 섬유아세포)가 어떻게 고정되고 면역염색되고 형광 또는 공초점 현미경으로 이미지되었는지를 설명합니다.

1 차 적인 섬모의 면역 형광 염색은 고품질 심상을 초래하는 상대적으로 간단한 절차입니다. 이러한 실험에서, 1차 섬모를 발현하는 섬유아세포는 위에서 설명한 프로토콜에 따라 형광 또는 공초점 현미경으로 고정, 면역염색, 이미지화되었다. 1차 실륨은 아세틸화된 α-tubulin 및 γ-tubulin을 사용하여 검출되었다. 1차 섬모의 평가는 다양한 수준에서 수행될 수 있으며 이와 관련하여 임의의 변화는 ?...

Watch this Scientific Journal Video about Simple Detection of Primary Cilia by Immunofluorescence at JoVE.com

Simple Detection of Primary Cilia by Immunofluorescence

1 차적인 섬모는 센티미터와 관련되었던 세포 외 구조물입니다. 면역 형광 염색에 의한 1 차 적인 섬모 검출은 매우 높은 품질의 심상을 초래하는 비교적 간단한 절차입니다. 이 프로토콜에서, 1 차적인 섬모를 표현하는 섬유아세포는 형광 또는 공초점 현미경으로 고정되고, 면역 염색되고, 심상화되었습니다.

면역 형광에 의한 1 차 적인 섬모의 간단한 검출

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to ...

이 프로토콜은 다양한 연구 노력 내에서 배양 된 세포에서 1 차 적인 섬모를 빠르고 쉽게 감지 할 수 있습니다. 방법은 1 차적인 섬모의 기초 바디에 영향을 미치는 무질서에서 이용될 수 있었습니다. 이 절차는 또한 파라핀 임베디드 조직에서 1 차적인 섬모를 염색하거나 형광이 필요한 그밖 실험을 위해 적용될 수 있습니다.22~22mm 커버 슬립을 오토클레이핑하고 실험을 위한 재료를 준비한다. FBS와 페니실린 연쇄 절제술을 해, 그리고 문체 매체를 실온으로 따뜻하게 합니다. 트립신 EDTA 및 PBS로 세포를 통과합니다.원고 의 방향에 따라 신선한 4 % 파라 포름 알데히드, 문화 매체 및 1 % 젤라틴 솔루션을 준비하십시오. 그런 다음 70 %에탄올로 라미나르 흐름 후드를 청소하고 필요한 모든 재료를 내부에 놓습니다. 파라포름알데히드는 매우 위험합니다.적절한 PPE는 항상 착용해야하며 화학 적 후드에서만 처리해야합니다. 원하는 세포를 70%로 성장한 후 인큐베이터에서 분리하여 라미나르 플로우 후드에 놓습니다. 배양 배지를 제거하고 PBS로 세포를 두 번 헹구는 다.배양 플라스크에 0.25%의 트립신 EDTA의 약 2밀리리터를 추가하고 5분 동안 섭씨 37도에서 배양합니다. 분리를 모니터링하기 위해 반전 된 현미경에서 세포를 주기적으로 확인하십시오. 세포가 분리되면 배양 배지의 10 밀리리터에서 부드럽게 다시 중단하여 단일 셀 서스펜션을 만들기 위해 조심스럽게 파이펫팅합니다.셀 서스펜션을 50밀리리터 원내 튜브로 옮기고 원심분리기를 5분 동안 200배 G'로 옮긴다. 상류제를 데칭한 다음 10 밀리리터의 배양 배지를 추가하고 펠릿을 부드럽게 재보냅니다. 셀 서스펜션의 마이크로리터 20개를 사용하여 일대일 볼륨 비율로 트라이팬 블루와 혼합하십시오.그런 다음 표준 혈중계를 사용하여 세포를 계산합니다. 6개의 웰 플레이트의 각 웰 안에 커버 슬립을 놓고 커버 슬립을 젤라틴 용액의 2밀리리터로 코팅하여 커버 슬립에 부착된 세포가 미끄러지는 데 도움이 됩니다. 젤라틴을 제거하고 접시가 몇 분 동안 공기 건조하게하십시오.씨앗 100, 000 각 우물에 섬유 아세포와 배양 배지의 두 밀리리터를 추가. 그런 다음 섭씨 37도, 이산화탄소 5%, 상대 습도 90%로 24시간 동안 세포를 배양합니다. 배양 후 세포를 치료하여 섬광을 유도한다.4%의 파라포름알데히드를 실온으로 데우고 목초지 파이펫, ABS, 폐기물 용기, 15밀리리터 원판 튜브, 마이크로 파이펫 및 팁을 준비합니다. 인큐베이터에서 세포를 가져 와서 실험실 벤치에 놓습니다. 각 우물에서 미디어를 제거하고 덮개가 미끄러져 둡깁니다.PBS의 두 밀리리터로 셀을 세 번 부드럽게 씻으십시오. 그런 다음 세포를 고치기 위해 각 웰에 4 %PFA의 2 밀리리터를 추가합니다. 상온에서 10분 동안 플레이트를 배양한 다음 PFA를 제거하고 PBS로 세척을 반복합니다.각 웰에 0.5%트리톤 X-100의 밀리리터 2개를 넣고 15분 동안 플레이트를 배양합니다. 그런 다음 PBS로 우물을 네 번 씻으시면 됩니다. 차단 용액을 만들려면 염소 세럼을 5분 동안 해동하고 PBS에서 1 대 20 비율로 희석합니다.이 솔루션의 마이크로리터 150개를 각 커버 슬립에 넣고 플레이트를 20분 동안 배양합니다. 1차 항체를 5분 동안 해동하고, 텍스트 원고에 설명된 대로 PBS에서 별도로 희석한다. 커버 슬립에서 차단 용액을 제거하고 두 항체 용액의 150 마이크로리터를 추가합니다.그런 다음 상온에서 60 분 동안 접시를 배양하십시오. 1차 항체를 제거하고 PBS의 2밀리리터로 커버 슬립을 세 번 부드럽게 씻습니다. CY3 양 항마우스 및 알렉사 플루오 488 염소 안티 토끼PBS에서 1 ~ 300 비율로 희석하고, 커버 슬립에 두 이차 항체 용액의 150 마이크로 리터를 추가합니다.PBS의 50 밀리리터에 10 마이크로리터를 희석하여 작동하는 DAPI 솔루션을 준비하고 이 희석제 2밀리리터를 커버 슬립에 추가합니다. 상온에서 어둠 속에서 5분 동안 접시를 배양합니다. 커버를 슬라이드에 놓기 전에 두 개의 바늘, 핀셋 및 장착 미디어를 준비하고 모든 슬라이드에 레이블을 지정합니다.우물에서 DAPI 용액을 제거하고 PBS로 세 번 세척합니다. 각 슬라이드에 마운팅 미디어를 한 방울 만 넣습니다. 바늘을 사용하여 우물 바닥에서 커버 슬립을 부드럽게 들어 올립니다.그런 다음 그것을 뒤집어 핀셋을 사용하여 장착 매체의 드롭 위에 부드럽게 놓습니다. 조심스럽게 거품을 제거합니다. 슬라이드를 빛으로부터 보호하고 섭씨 4도에서 밤새 보관하십시오.다음 날에는 높은 배율로 형광 또는 공초점 현미경을 사용하여 1 차 적인 섬모를 시각화합니다. 이 프로토콜은 면역스테인을 고치고, 1차 섬모를 발현하는 다양한 세포주를 이미지화하는 데 사용되었다. 마우스 근세포가 다양한 용량에서 이온화 방사선에 노출되었을 때, 더 높은 용량이 다중 1차 섬모의 출현을 유도하는 것으로 나타났다.낮은 복용량 단일 기본 섬모의 출현 귀 착되 게 하는 동안. 유사하게, 인간 폐 섬유아세포가 저용량으로 방사되었을 때, 단일 1차 섬모는 면역형광에 의해 검출되었다. 1 차적인 섬모에 있는 증가는 신진 대사 스트레스가 1 차적인 섬모의 사건에 영향을 미친다는 것을 보여주는 굶주린 마우스 배아 섬유아세포에서 관찰되었습니다.피부 섬유아세포가 120 나노몰러 독소루비신으로 치료되었을 때, 그들은 단일 1 차실을 표현했다. 더 높은 복용량 여러 기본 섬모의 모양을 유도. 1.25 나노 몰라 탁솔로 치료 된 섬유 아세포도 단일 기본 실륨을 표현했다.그러나 다중 섬모는 더 높은 복용량으로 처리 후에 검출되지 않았습니다. 이 프로토콜을 열 때 모든 PPE 규정을 따라야 합니다. 선택의 세포주의 배양 요구를 명심하고 신중하게 항체를 선택하십시오.이 절차는 세포가 복구할 수 없기 때문에 다른 절차를 직접 따를 수 없습니다. 대신 병렬 실험을 프로그래밍해야 합니다.

simple-detection-of-primary-cilia-by-immunofluorescence

Research

JoVE Journal

Biology

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxy&#64258;uorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.

기본 조직의 두 Thymidine의 Analogues의 Immunofluorescent 검색 (CldU 및 이두)

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

연구

생물학

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

Zymography의 기능 매트릭스 Metalloproteinases의 감지

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

엘리사에 의해 네이티브 손상 Nucleosomes에 포스트 translational 수정의 감지

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

형광에 의한 바이러스성 RNA의 탐지<em&gt; 현장에서</em&gt; 하이브리드화 (물고기)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Internal organs such as the heart, brain, and gut develop left-right (LR) asymmetries that are critical for their normal functions1. Motile cilia are involved in establishing LR asymmetry in vertebrate embryos, including mouse, frog, and zebrafish2-6. These 'LR cilia' generate asymmetric fluid flow that is necessary to trigger a conserved asymmetric Nodal (TGF-&beta; superfamily) signaling cascade in the left lateral plate mesoderm, which is thought to provide LR patterning information for developing organs7. Thus, to understand mechanisms underlying LR patterning, it is essential to identify genes that regulate the organization of LR ciliated cells, the motility and length of LR cilia and their ability to generate robust asymmetric flow.
In the zebrafish embryo, LR cilia are located in Kupffer's vesicle (KV)2,4,5. KV is comprised of a single layer of monociliated epithelial cells that enclose a fluid-filled lumen. Fate mapping has shown that KV is derived from a group of ~20-30 cells known as dorsal forerunner cells (DFCs) that migrate at the dorsal blastoderm margin during epiboly stages8,9. During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo10,11. The ability to identify and track DFCs&mdash;in combination with optical transparency and rapid development of the zebrafish embryo&mdash;make zebrafish KV an excellent model system to study LR ciliated cells.
Interestingly, progenitors of the DFC/KV cell lineage retain cytoplasmic bridges between the yolk cell up to 4 hr post-fertilization (hpf), whereas cytoplasmic bridges between the yolk cell and other embryonic cells close after 2 hpf8. Taking advantage of these cytoplasmic bridges, we developed a stage-specific injection strategy to deliver morpholino oligonucleotides (MO) exclusively to DFCs and knockdown the function of a targeted gene in these cells12. This technique creates chimeric embryos in which gene function is knocked down in the DFC/KV lineage developing in the context of a wild-type embryo. To analyze asymmetric fluid flow in KV, we inject fluorescent microbeads into the KV lumen and record bead movement using videomicroscopy2. Fluid flow is easily visualized and can be quantified by tracking bead displacement over time.
Here, using the stage-specific DFC-targeted gene knockdown technique and injection of fluorescent microbeads into KV to visualize flow, we present a protocol that provides an effective approach to characterize the role of a particular gene during KV development and function.

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer&#039;s Vesicle

Cilia-generated fluid flow in Kupffer&#x2019;s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.

Kupffer의 소포의 유전자 기능 및 속눈썹 생성 된 유체 흐름의 시각화 분석

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

라이브의 검출<em&gt; 대장균</em&gt; O157 : PMA-qPCR에 의한 H7 셀

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

공 초점 현미경으로 리간드 - 활성화 된 G 단백질 결합 수용체 내재화 검출

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...