Name: 5-ethynyl-2'-deoxyuridine/phospho-histone h3 dual-labeling protocol for cell cycle progression analysis in drosophila neural stem cells
Uploaded: 2021-05-04T15:00:00
Description: Watch this Scientific Journal Video about 5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells at JoVE.com

This work was supported by funding from the Swiss National Science Foundation (project grant 31003A_173188; www.snf.ch) and the University of Bern (www.unibe.ch) to BS. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

1. Preparation of reagents and stocks for Click-it EdU assay
NOTE: Refer to the Table of Materials and Table 1 for details about the kit and reagents supplied with the kit.
<ol>
	<li>Bring the vials to room temperature before preparing the solutions.</li>
	<li>Prepare 10 mM EdU (component A) stock solution by adding 2 mL of dimethylsulfoxide (DMSO, component C). Mix well and store at -20 &#176;C.</li>
	<li>Prepare a working solution of Alexa Fluor 647-azide ...

<ol>
	<li>Harashima, H., Dissmeyer, N., Schnittger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+control+across+the+eukaryotic+kingdom.">Cell cycle control across the eukaryotic kingdom.</a> Trends in Cell Biology. 23 (7), 345-356 (2013).</li><li>Vermeulen, K., Van Bockstaele, D. R., Berneman, Z. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+cycle:+a+review+of+regulation,+deregulation+and+therapeutic+targets+in+cancer.">The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer.</a> Cell Proliferation. 36 (3), 131-149 (2003).</li><li>Pardee, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+restriction+point+for+control+of+normal+animal+cell+proliferation.">A restriction point for control of normal animal cell proliferation.</a> Proceedings of the National Academy of the Sciences of the United States of America. 71 (4), 1286-1290 (1974).</li><li>Gogendeau, 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=Aneuploidy+causes+premature+differentiation+of+neural+and+intestinal+stem+cells.">Aneuploidy causes premature differentiation of neural and intestinal stem cells.</a> Nature Communications. 6, 8894 (2015).</li><li>Barnum, K. J., O'Connell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+regulation+by+checkpoints.">Cell cycle regulation by checkpoints.</a> Methods in Molecular Biology. 1170, 29-40 (2014).</li><li>Gratzner, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibody+to+5-bromo-+and+5-iododeoxyuridine:+A+new+reagent+for+detection+of+DNA+replication.">Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication.</a> Science. 218 (4571), 474-475 (1982).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proceedings of the National Academy of the Sciences of the United States of America. 105 (7), 2415-2420 (2008).</li><li>Kim, J. Y., 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+value+of+phosphohistone+H3+as+a+proliferation+marker+for+evaluating+invasive+breast+cancers:+A+comparative+study+with+Ki67.">The value of phosphohistone H3 as a proliferation marker for evaluating invasive breast cancers: A comparative study with Ki67.</a> Oncotarget. 8 (39), 65064-65076 (2017).</li><li>Homem, C. C., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+neuroblasts:+a+model+for+stem+cell+biology.">Drosophila neuroblasts: a model for stem cell biology.</a> Development. 139 (23), 4297-4310 (2012).</li><li>Daul, A. L., Komori, H., Lee, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EdU+(5-ethynyl-2'-deoxyuridine)+labeling+of+Drosophila+mitotic+neuroblasts.">EdU (5-ethynyl-2'-deoxyuridine) labeling of Drosophila mitotic neuroblasts.</a> Cold Spring Harbor Protocols. 2010 (7), 5461 (2010).</li><li>Chippalkatti, R., Egger, B., Suter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mms19+promotes+spindle+microtubule+assembly+in+Drosophila+neural+stem+cells.">Mms19 promotes spindle microtubule assembly in Drosophila neural stem cells.</a> PLoS Genetics. 16, 1008913 (2020).</li><li>Daul, A. L., Komori, H., Lee, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescent+staining+of+Drosophila+larval+brain+tissue.">Immunofluorescent staining of Drosophila larval brain tissue.</a> Cold Spring Harbor Protocols. (7), (2010).</li><li>Mollinari, C., Lange, B., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miranda,+a+protein+involved+in+neuroblast+asymmetric+division,+is+associated+with+embryonic+centrosomes+of+Drosophila+melanogaster.">Miranda, a protein involved in neuroblast asymmetric division, is associated with embryonic centrosomes of Drosophila melanogaster.</a> Biology of the Cell. 94 (1), 1-13 (2002).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Nag, R. N., Niggli, S., Sousa-Guimaraes, S., Vazquez-Pianzola, P., Suter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mms19+is+a+mitotic+gene+that+permits+Cdk7+to+be+fully+active+as+a+Cdk-activating+kinase.">Mms19 is a mitotic gene that permits Cdk7 to be fully active as a Cdk-activating kinase.</a> Development. 145 (2), (2018).</li><li>Cabernard, C., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+neuroblast+lineages+within+intact+larval+brains+in+Drosophila.">Live imaging of neuroblast lineages within intact larval brains in Drosophila.</a> Cold Spring Harbor Protocols. 2013 (10), (2013).</li><li>Hartenstein, V., Spindler, S., Pereanu, W., Fung, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+Drosophila+larval+brain.">The development of the Drosophila larval brain.</a> Advances in Experimental Medicine and Biology. 628, 1-31 (2008).</li><li>Hebar, A., Rutgen, B. C., Selzer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NVX-412,+a+new+oncology+drug+candidate,+induces+S-phase+arrest+and+DNA+damage+in+cancer+cells+in+a+p53-independent+manner.">NVX-412, a new oncology drug candidate, induces S-phase arrest and DNA damage in cancer cells in a p53-independent manner.</a> PLoS One. 7, 45015 (2012).</li><li>Wyllie, F. 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=S+phase+cell-cycle+arrest+following+DNA+damage+is+independent+of+the+p53/p21(WAF1)+signalling+pathway.">S phase cell-cycle arrest following DNA damage is independent of the p53/p21(WAF1) signalling pathway.</a> Oncogene. 12 (5), 1077-1082 (1996).</li><li>Xu, X., 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=Inhibition+of+DNA+replication+and+induction+of+S+phase+cell+cycle+arrest+by+G-rich+oligonucleotides.">Inhibition of DNA replication and induction of S phase cell cycle arrest by G-rich oligonucleotides.</a> Journal of Biological Chemistry. 276 (46), 43221-43230 (2001).</li><li>Duronio, 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=Developing+S-phase+control.">Developing S-phase control.</a> Genes &amp; Development. 26 (8), 746-750 (2012).</li><li>Turrero Garcia, M., Chang, Y., Arai, Y., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-phase+duration+is+the+main+target+of+cell+cycle+regulation+in+neural+progenitors+of+developing+ferret+neocortex.">S-phase duration is the main target of cell cycle regulation in neural progenitors of developing ferret neocortex.</a> Journal of Comparative Neurology. 524 (3), 456-470 (2016).</li><li>Pereira, P. 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=Quantification+of+cell+cycle+kinetics+by+EdU+(5-ethynyl-2'-deoxyuridine)-coupled-fluorescence-intensity+analysis.">Quantification of cell cycle kinetics by EdU (5-ethynyl-2'-deoxyuridine)-coupled-fluorescence-intensity analysis.</a> Oncotarget. 8 (25), 40514-40532 (2017).</li><li>Sakaue-Sawano, 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=Visualizing+spatiotemporal+dynamics+of+multicellular+cell-cycle+progression.">Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.</a> Cell. 132 (3), 487-498 (2008).</li><li>Zielke, N., 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=Fly-FUCCI:+A+versatile+tool+for+studying+cell+proliferation+in+complex+tissues.">Fly-FUCCI: A versatile tool for studying cell proliferation in complex tissues.</a> Cell Reports. 7 (2), 588-598 (2014).</li><li>Shen, Y., Vignali, P., Wang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+profiling+cell+cycle+by+flow+cytometry+using+concurrent+staining+of+DNA+and+mitotic+markers.">Rapid profiling cell cycle by flow cytometry using concurrent staining of DNA and mitotic markers.</a> Bio-protocol. 7 (16), 2517 (2017).</li><li>Berger, 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=FACS+purification+and+transcriptome+analysis+of+drosophila+neural+stem+cells+reveals+a+role+for+Klumpfuss+in+self-renewal.">FACS purification and transcriptome analysis of drosophila neural stem cells reveals a role for Klumpfuss in self-renewal.</a> Cell Reports. 2 (2), 407-418 (2012).</li><li>Harzer, H., Berger, C., Conder, R., Schmauss, G., Knoblich, J. 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EdU incorporation and its subsequent &#39;click&#39; reaction with cell-permeable azide presents practical advantages of this technique over the BrdU method used earlier7. However, this reaction is catalyzed by Cu(I) ions, and several dyes may be unstable in the presence of this copper catalyst, as is clearly advised by the Click-it EdU kit manufacturer. When immunostaining experiments had been performed after executing the EdU detection step, the expected signal intensity was not observed with th...

The cell division cycle comprises a G1 phase (first gap phase), a replicative/S-phase, a G2 (second gap phase), and an M (mitotic) phase. Passing through these phases, the cell undergoes dramatic changes in cellular transcription, translation, and re-organization of cytoskeletal machinery1,2. In response to developmental and environmental cues, cells may temporarily cease to divide and become quiescent (G0) or differentiate and permanently cease to divide3. Other scenarios, such as DNA damage, may cause premature differentiation or apoptosis3,4. Response to such cues is mediated by cell cycle checkpoints, which act as a surveillance system to ensure the integrity of essential cellular processes before the cell commits to the next phase of the division cycle5. Therefore, studies on genes regulating DNA replication, checkpoints, and mitotic machinery need to analyze possible cell cycle progression defects that may occur in mutant cells or upon siRNA knockdown of these genes. Additionally, such analyses may be employed to test overall cell health as well as cellular responses to drug treatment.
5-bromo-2&#39;-deoxyuridine (BrdU) is a thymidine analog that is incorporated into DNA during replication6. This method was used extensively to identify cells in S-phase. However, the cells are then subjected to harsh DNA denaturation procedures to allow detection of BrdU through the use of anti-BrdU antibodies6. This harsh treatment may damage cellular epitopes and prevent further characterization of the sample through immunostaining. EdU incorporation and subsequent detection by a copper-catalyzed, &#39;click reaction&#39; with small, cell-permeable, fluorescently tagged azide dyes eliminates the need for harsh denaturation procedures7. This method, therefore, emerged as a more practical alternative to BrdU incorporation.
Further, pH3 has been described as a reliable marker for mitotic/M phase cells8. Histone H3 is a DNA-associated core histone protein that becomes phosphorylated in around the late G2 phase to early M phase and is de-phosphorylated toward the end of anaphase8. Several commercial antibodies can be used to detect pH3 using standard immunostaining protocols. Dual-staining of EdU and pH3 would therefore enable the detection of cells in S-phase as well as M-phase. Additionally, cells in the G1 and early G2 phase would not stain positively for either of the markers.
Drosophila neural stem cells or neuroblasts (NBs) offer a well-characterized stem cell model wherein cells divide asymmetrically to produce one identical self-renewing NB and a ganglion mother cell (GMC), which is fated for differentiation9. Additionally, several genetic tools and NB-specific antibodies make this system suitable for genetic manipulation and live-cell imaging. Consequently, several studies have utilized NBs to study genes regulating asymmetric divisions and cell fate determination9. Distinct populations of NBs exist in the central brain (CB) and the optic lobe (OL) of the larval brain9; CB NBs were used for the current study. These third instar larval CB NBs are large cells that are also suitable for studying factors regulating mitotic spindle assembly. A protocol to analyze cell cycle progression defects would be a vital tool in such studies.
Protocols published earlier employed commercial kits, such as Click-iT EdU Alexa Fluor Cell Proliferation Kit, which provide several reaction components and azide dyes tagged with a variety of Alexa Fluor dyes for EdU incorporation and detection10. However, the reagents supplied with such kits are not compatible with some fluorescent tags often used with secondary antibodies. This EdU detection kit (Click-iT EdU Alexa Fluor Cell Proliferation Kit supplied with Alexa Fluor 647-conjugated azide dye) was tested in Drosophila third instar larval NBs, and co-staining was attempted with antibodies against pH3 and Miranda, a marker for NBs. Further, Alexa Fluor 568- or Cy3-tagged secondary antibodies were used for detection of Miranda labeling on the plasma membrane of NBs11. However, the expected signal intensity and staining pattern (unpublished results) were not observed with these secondary antibodies when immunostaining was performed after EdU detection.
For EdU incorporation, the protocol described by Daul and colleagues required feeding of the larvae with Kankel-White medium mixed with EdU and bromophenol blue (BPB)10. The larvae fed on the EdU and BPB-spiked food, which could be seen by its blue color upon ingestion in the larval gut. Although this method was used for EdU incorporation in Mms19 loss-of-function (Mms19P) third instar larvae, the Mms19P larvae apparently did not feed as hardly any blue color was detected in the larval gut (unpublished results). The Mms19P larvae show drastic developmental deformities and eventually arrest in the third instar stage. This may somehow affect the feeding behavior of the third instar larvae and render the EdU-feeding protocol unsuitable for such cases.
After studying the available literature and working extensively on the standardization of essential steps, an alternative approach was proposed for EdU/pH3 dual-labeling in Drosophila NBs, which does not require feeding EdU to larvae. A previous study employed dual EdU/pH3 staining to analyze the cell cycle in NBs, but did not present a detailed protocol4. This presents an unnecessary hurdle for labs trying to implement this method. Furthermore, evaluating the compatibility of various reagents with the EdU kit and performing further optimization can be a time-consuming process. This paper presents a step-by-step protocol that covers EdU incorporation in dissected larval brains and immunostaining with anti-pH3 antibodies, followed by confocal microscopy and image analysis to allocate NBs to four distinct categories: G0/G1 phase, S phase, S&#62;G2/M (progression from S to G2/M), and M phase. The steps that need optimization are outlined and tips provided for image analysis of large datasets. Additionally, the EdU/pH3 readout in wild-type NBs is analyzed and compared with Mms19P&#160;NBs, which were recently reported to show a cell cycle delay11.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>fly stocks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P{EPgy2}Mms19EY00797/TM3, Sb1 Ser1 (Mms19p)</td>
			<td>Bloomington stock center</td>
			<td>#15477</td>
			<td>P-element insertion in the third exon of Mms19</td>
		</tr>
		<tr>
			<td>&#160;+; Mms19::eGFP, Mms19p</td>
			<td>Generated in house</td>
			<td></td>
			<td>eGFP-tagged Mms19 protein expressed in Mms19p background</td>
		</tr>
		<tr>
			<td>w1118</td>
			<td>Bloomington stock center</td>
			<td>#3605</td>
			<td>wild-type stock (w;+;+)</td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td></td>
			<td></td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>Rat anti-Miranda</td>
			<td>Abcam</td>
			<td>Ab197788</td>
			<td>1/250</td>
		</tr>
		<tr>
			<td>Rabbit anti-pH3</td>
			<td>Cell Signaling</td>
			<td>9701</td>
			<td>1/200</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rat Cy3</td>
			<td>Jackson Immuno</td>
			<td>112-165-167</td>
			<td>1/150</td>
		</tr>
		<tr>
			<td>Goat anti-Rat Alexa Fluor 568</td>
			<td>Invitrogen</td>
			<td>A11077</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A27034</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Reagent/Kit</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua Poly/Mount mounting medium</td>
			<td>Polysciences Inc</td>
			<td>18606-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Click-it EdU incorporation kit, Alexa Flour 647</td>
			<td>Thermo Fischer Scientific</td>
			<td>C10340</td>
			<td></td>
		</tr>
		<tr>
			<td>Schneider&#8217;s Drosophila medium</td>
			<td>Thermo Fischer Scientific</td>
			<td>21720-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA) fraction V</td>
			<td>Merck</td>
			<td>10735078001</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fischer Scientific</td>
			<td>9002-93-1</td>
			<td>non-ionic detergent</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji (Imagej)</td>
			<td>https://imagej.net/Fiji</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite (LAS X)</td>
			<td>Leica microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PRISM</td>
			<td>Graph pad software</td>
			<td>Version 5</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft office</td>
			<td>2016</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP8 laser scanning confocal microscope with 63x oil-immersion, 1.4 NA Plan-apochromat objective</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua Poly/Mount mounting medium</td>
			<td></td>
			<td></td>
			<td>water-soluble, non-fluorescing mounting medium</td>
		</tr>
		<tr>
			<td>Pyrex 3 or 9 depression glass spot plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman filter paper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

5-ethynyl-2'-deoxyuridine/phospho-histone h3 dual-labeling protocol for cell cycle progression analysis in drosophila neural stem cells

In vivo cell cycle progression analysis is routinely performed in studies on genes regulating mitosis and DNA replication. 5-Ethynyl-2'-deoxyuridine (EdU) has been utilized to investigate replicative/S-phase progression, whereas antibodies against phospho-histone H3 have been utilized to mark mitotic nuclei and cells. A combination of both labels would enable the classification of G0/G1 (Gap phase), S (replicative), and M (mitotic) phases and serve as an important tool to evaluate the effects of mitotic gene knockdowns or null mutants on cell cycle progression. However, the reagents used to mark EdU-labelled cells are incompatible with several secondary antibody-fluorescent tags. This complicates immunostaining, where primary and tagged secondary antibodies are used to mark pH3-positive mitotic cells. This paper describes a step-by-step protocol for the dual-labeling of EdU and pH3 in Drosophila larval neural stem cells, a system utilized extensively to study mitotic factors. Additionally, a protocol is provided for image analysis and quantification to allocate labeled cells in 3 distinct categories, G0/G1, S, S&gt;G2/M (progression from S to G2/M), and M phases.

The bi-lobed Drosophila third instar larval brain has been utilized as a model system to study fundamental cellular and developmental processes9. The focus of the current study was to present a protocol for analysis of cell cycle progression in EdU- and pH3-labeled NBs of the CB region (Figure 1). The CB NBs are sub-divided into type I and type II, and they display the characteristic asymmetric division pattern9. Each type I NB divisio...

Watch this Scientific Journal Video about 5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells at JoVE.com

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells

Cell cycle analysis with 5-ethynyl-2'-deoxyuridine (EdU) and phospho-histone H3 (pH3) labeling is a multi-step procedure that may require extensive optimization. Here, we present a detailed protocol that describes all steps for this procedure including image analysis and quantification to distinguish cells in different cell cycle phases.

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells

In vivo cell cycle progression analysis is routinely performed in studies on genes regulating mitosis and DNA replication. 5-Ethynyl-2'-deoxyuridine (EdU) ...

In this protocol, we not only provide a detailed explanation of each step of EDU incorporation and immunostaining, but also explain image acquisition, image processing, and provide some tips for analyzing large datasets. This technique allows for the allocation of cells in G1, S, and M phases. This will be particularly useful for cell cycle analysis on studies on non mutations affecting mitotic genes.We would expect people to struggle for two to three months if they do not have prior knowledge or experience with this technique. To begin, add Schneider's dissection medium or SM to two consecutive depressions of a gloss spot plate and add PBS to the subsequent depression. Using a pair of forceps, pick wandering third instar larvae and place them on a tissue ratted with PBS to clean off fly food residue.Place the larva in the depression with PBS followed by the consecutive depression containing SM.Next under a dissection microscope, use fine forceps to remove three quarters of the lower body of the larva. Then gently grab the larval mouth hooks with one pair of forceps and hold the cuticle of the other end with the other pair of forceps. Turn the larval head inside out by pushing the mouth hooks inward and simultaneously peeling off the tissue at the other end.Observe the larval brain with attached imaginal discs. Remove other tissues attached to the brain and transfer the brain to the subsequent consecutive depression with SM.Add 100 microliters of 100 micromolar EDU SM solution to another depression on the Pyrex plate and incubate approximately 5 to 10 dissected brains in this solution for 2 hours at 25 degrees Celsius. After fixation and immunostaining remove PBST and add the EDU detection cocktail to the dissected brains.Then incubate them at room temperature in the dark on a neutater. After 30 minutes, wash the samples twice with PBST for 10 minutes per wash in the dark. For DNA staining, prepare 5 micrograms per milliliter Hoechst 33342 solution.Remove PBST after the second wash and add 500 microliters of the Hoechst solution to the dissected brains, then incubate them in the dark. After 10 minutes, remove the Hoechst and wash the samples with PBST for 10 minutes. Tap the tube onto the lab bench and let the brains settle down.Remove the PBST from the tube, leaving behind 50 to 100 microliters. Cut the end of a 200 microliter micropipet tip and use it to transfer the brains onto a clean glass slide. Remove access PBST from the slide by blotting it with filter paper strips.Do not let the filter paper touch the brains. Put a drop of a water soluble, non fluorescing mounting medium onto the brains and orient the brains such that the ventral nerve cord faces the slide and the lobes face upwards. Arrange the brains in a single file so that it is easier to image the brains serially.Gently place a cover slip on top of the brain and incubate the slide overnight at 2 to 6 degrees Celsius. From the acquisition software select the 63 times objective. Put a drop of immersion oil on the cover slip just above the mounted brains to make it easier to locate the tissue through the eyepiece.Using the dappy Hoechst 33342 channel, find the brain through the eyepiece and then switch to the acquisition mode in the software. Set up 4 channels to image Hoechst 33342, Alexa floor 4 8 8, Alexa floor 5 6, 8, and Alexa floor 6 4 7. Use the dye assistant tool to automatically set up the selected dyes excitation lasers and emission filters.Set the field of view, such that it encompasses the entire brain lobe. Image the entire volume of the brain lobe by acquiring this tax spaced 0.8 micron apart. Store all images from an imaging session in a dot L I F library format.For image analysis, open Fiji, then drag, and drop the LIF files into Fiji. Select data browser from the stack viewing tab and use virtual stack from the memory management tab in the bio formats plugin. Observe the multi-channel image displayed in image J.Change the color of the channels from the menu bar, using image, color, and channels tool.And monitor the Miranda labeled neuroblasts, which appear as large round cells in the central brain region. Draw a region of interest using the ellipse tool over each neuroblast to avoid counting the neuroblast twice. From the image J menu bar, select analyze, tools, and ROI manager.Mark all neuroblasts in the current Z section and press T after marking each cell. Once all neuroblasts in the current Z-section are marked, change the channels to pH3 and EDU and manually count the number of EDU positive neuroblasts, pH3 positive neuroblasts, EDU and pH3 positive neuroblasts, and neuroblasts not staining for either marker. Search for neuroblasts and subsequent Z sections, delete old ROIs and add new ROIs to count neuroblasts in different stacks.Prepare a spreadsheet and calculate percentages of neuroblasts present in all four categories for each lobe. Prepare a bar graph using the spreadsheets software, showing the pools data for percentages of neuroblasts in each category. Central brain neuroblasts from third instar larval brains marked with Miranda, EDU, and pH3 are shown here.Cells were allocated to G1/G0, S, SG2/M, and M phases. In EDU pH3 analysis, a significantly higher proportion of MMS19, loss of function neuroblasts was found in the M phase compared to wild type neuroblasts. MMS19:eGFP expression in the MMS19 loss of function background rescued the proportion of cells in M phase to wild type levels.It is important not to use damaged brains for subsequent steps. And ED solution should be prepared freshly before starting dissections. This asset can be used as a quick initial screening to identify defects in specific cell cycle phases.This could then be followed up by a more detailed analysis of a particular phase.

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