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

This work was supported by the Ministry of Defence of the Czech Republic -&#160;Long-term organization development plan Medical Aspects of Weapons of Mass Destruction of the Faculty of Military Health Sciences, University of Defence; the Ministry of Education, Youth and Sport, Czech Republic (Specific Research Project No: SV/ FVZ201703) and PROGRES Q40/06. Thanks also to Daniel Diaz for his kind assistance in English language revision.

1. Preparation of culture media, solutions, and dishes
<ol>
	<li>Autoclave the coverslips (22 x 22 mm). Prepare 6 well plates. Thaw fetal bovine serum (FBS) and antibiotic penicillin/streptomycin and warm the culture medium to room temperature (RT). Use trypsin-EDTA (0.25%) and 1x PBS (phosphate buffered saline with calcium and magnesium) to passage the cells.</li>
	<li>Prepare fresh 4% paraformaldehyde (PFA) in dH2O (800 mg of PFA in 20 mL of dH2O). The PFA must be freshly prepared for each exper...

<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>

Several authors have described diverse methods for the detection of primary cilia, sometimes also describing various fixation methods that can affect their detection6,20,21,22. Regardless, it is difficult to find a complete and straightforward protocol for detection. The ready availability of such a method would undoubtedly be of great assistance to the study of primary cilia investigation, esp...

Primary cilia are sensory, solitary, membrane-bound, nonmotile structures associated with the cell&#8217;s mother centriole. Primary cilia are found on most vertebrate cells with the exception of red blood cells, adipocytes1, and hepatocytes2. Primary cilia are formed as an elongated axoneme composed by microtubules, whose main component is &#945;-tubulin. The axoneme grows from the basal body, which is structured from &#947;-tubulin. The length of the primary cilia varies between 2&#8211;10 &#181;m; however, its dimensions can change during glycylation, starvation, hypoxia, cytotoxic stress, or after exposure to ionizing radiation3,4,5,6,7. Usually, cells have only one primary cilium, which is involved in morphogenesis and cell signalling pathways important for cell proliferation and differentiation8,9.
Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases, and resorbed before entering mitosis in a process associated with tubulin deacetylation mediated by HDAC6 (histone deacetylase 6)10. The exact moment of primary cilia resorption depends upon cell type and the expression of genes directly involved in this process, such as Aurora A, Plk1, TcTex-111,12,13. Depending on the cell type, the primary cilia express different types of receptors, ion channels, and active signalling pathways. These include the most important signalling receptors affecting proliferation and survival, EGFR, PDGFR, and FGFR. Also included are some of the signalling pathways that may affect the function of one or more organs, including Hedgehog, Notch, and Wnt. Thanks to these receptors and signalling pathways, the primary cilia also perform a chemosensory function. This function allows primary cilia to detect specific ligands for Notch, hormones, and biologically active substances such as serotonin or somatostatin. Other specific functions exhibited by primary cilia of different lengths include reaction to changes in temperature, gravity, and osmolality14.
Primary cilia can be visualized through various methods, such as live visualization, transmission electron microscopy, 3D imaging, or by software for the automatic detection of primary cilia5,15,16,17. However, these methods are highly specialized and ongoing research needs basic, fast, and easy methods for staining primary cilia in every stage of research. Described is an easy and useful method for the detection of primary cilia in cultured cells.

<table border="0" cellpadding="0" cellspacing="0" style="width:1301px;" width="1301">
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	<tbody>
		<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

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to mitosis. Primary cilia can be visualized with highly sophisticated methods, including transmission electron microscopy, 3D imaging, or using software for the automatic detection of primary cilia. However, immunofluorescent staining of primary cilia is needed to perform these methods. This publication describes a protocol for the easy detection of primary cilia in vitro by staining acetylated alpha tubulin (axoneme) and gamma tubulin (basal body). This immunofluorescent staining protocol is relatively simple and results in high-quality images. The present protocol describes how four cell lines (C2C12, MEF, NHLF, and skin fibroblasts) expressing primary cilia were fixed, immunostained, and imaged with a fluorescent or confocal microscope.

The immunofluorescent staining of primary cilia is a relatively simple procedure that results in high-quality images. In these experiments, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope following the protocol described above. The primary cilium was detected using acetylated &#945;-tubulin and &#947;-tubulin. The evaluation of primary cilia can be performed on various levels and any change in this regard can be linked to exposure to ionizing radiation, c...

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

Simple Detection of Primary Cilia by Immunofluorescence

Primary cilia are extracellular structures associated with the centriole. Primary cilia detection by immunofluorescent staining is a relatively simple procedure that results in extremely high-quality images. In this protocol, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope.

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

This protocol allows for quick and easy detection of primary cilia in cultured cells, within a variety of research endeavors. The method could be utilized in disorder that affect the basal bodies of primary cilia. This procedure can also be applied for staining primary cilia in paraffin embedded tissue or for other experiments where fluorescence is needed.Begin by autoclaving 22 by 22 millimeter cover slips, and preparing materials for the experiment. Thaw FBS and penicillin streptomycin, and warm the culture medium to room temperature. Passage the cells with trypsin EDTA and PBS.Prepare fresh 4%paraformaldehyde, culture medium, and 1%gelatin solution, according to manuscript directions. Then clean the laminar flow hood with 70%ethanol, and place all required materials inside. Paraformaldehyde is extremely hazardous.Proper PPE has to be worn at all times, and it should only be handled in a chemical hood. After growing the desired cells to 70%confluence remove them from the incubator, and place them in the laminar flow hood. Remove the culture medium and rinse the cells twice with PBS.Add about two milliliters of 0.25%trypsin EDTA to the culture flask, and incubate it at 37 degrees Celsius for five minutes. Check the cells periodically on the inverted microscope to monitor detachment. When the cells have detached gently resuspend them in 10 milliliters of culture medium, pipetting carefully to create a single cell suspension.Transfer the cell suspension to a 50 milliliter conical tube and centrifuge it at 200 times G'for five minutes. Decant the supernatant then add 10 milliliters of culture medium, and gently resuspend the pellet. Take 20 microliters of the cell suspension and mix it with trypan blue at a one-to-one volume ratio.Then count the cells using standard hemocytometry. Place a cover slip inside each well of a six well plate, and coat the cover slip with two milliliters of the gelatin solution, which will help the cells attached to the cover slip. Remove the gelatin and let the plate air-dry for a few minutes.Seed 100, 000 fibroblasts into each well and add two milliliters of culture medium. Then incubate the cells for 24 hours at 37 degrees Celsius, five percent carbon dioxide, and 90%relative humidity. After the incubation treat the cells to induce ciliation.Warm the 4%paraformaldehyde to room temperature, and prepare a pasture pipettes, ABS, a waste container, 15 milliliter conical tubes, micro pipettes, and tips. Take the cells from the incubator, and place them on the lab bench. Remove media from each well, leaving the cover slip.Gently wash the cells three times with two milliliters of PBS. Then add two milliliters of the 4%PFA into each well to fix the cells. Incubate the plate for 10 minutes at room temperature, then remove the PFA, and repeat the washes with PBS.Add two milliliters of 0.5%Triton X-100 to each well, and incubate the plate for 15 minutes. Then wash the wells four times with PBS. To make the blocking solution, thaw a goat serum for five minutes, and dilute it in PBS at a one to 20 ratio.Add 150 microliters of this solution to each cover slip, and incubate the plate for 20 minutes. Thaw the primary antibodies for five minutes, and dilute them separately in PBS, as described in the text manuscript. Remove the blocking solution from the cover slips and add 150 microliters of both antibody solutions.Then incubate the plate for 60 minutes at room temperature. Remove the primary antibodies and gently wash the cover slips three times with two milliliters of PBS. Dilute Cy3 Sheep Anti-Mouse and Alexa fluor 488 Goat anti-Rabbit in PBS, at a one to 300 ratio, and add 150 microliters of both secondary antibody solutions to the cover slip.Prepare a working DAPI solution by diluting 10 microliters of the stock in 50 milliliters of PBS, and add two milliliters of this dilution to the cover slips. Incubate the plate for five minutes in the dark, at room temperature. Before placing the cover slips on the slides, prepare two needles, tweezers, and mounting media, and label all the slides.Remove the DAPI solution from the wells, and wash them three times with PBS. Put one drop of mounting media on each slide. Use the needle to gently lift the cover slip from the well's bottom.Then flip it and gently place it over the drop of mounting media using the tweezers. Carefully remove any bubbles. Protect the slides from light and store them overnight at four degrees Celsius.On the next day use a fluorescent or confocal microscope with a high magnification to visualize the primary cilia. This protocol was used to fix immunostain, and image various cell lines expressing primary cilia. When mouse myoblasts were exposed to ionizing radiation at various doses, it was found that higher doses induce the appearance of multiple primary cilia.While low doses resulted in the appearance of single primary cilia. Similarly, when human lung fibroblasts were radiated at a low dose, single primary cilia were detected by immunofluorescence. An increase in primary cilia was observed in mouse embryonic fibroblasts that were starved, demonstrating that metabolic stress affects the incidents of primary cilia.When skin fibroblasts were treated with 120 nanomolar doxorubicin, they expressed a single primary cilium. Higher doses induce the appearance of multiple primary cilia. Fibroblasts treated with 1.25 nanomolar Taxol also expressed a single primary cilium.But multiple cilia were not detected after treatment with higher doses. When opening this protocol make sure to follow all PPE regulations. Keep in mind the culture needs of your cell line of choice and chose your antibodies carefully.This procedure cannot be directly followed by other procedures, since the cells are irretrievable. Instead, parallel experiments should be programmed.

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.

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

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.

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.

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.

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.

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.

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

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.

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.

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.

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