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 (component B) by adding 70&#956;L of DMSO (component C). Mix well and store at -20 &#176;C.</li>
	<li>Prepare a 1x solution of Click-iT EdU reaction buffer (component D) by mixing 4 mL of this solution with 36 mL of deionized water. Store the remaining solution at 2-6 &#176;C.</li>
	<li>Make a 10x stock (200 mg/mL) of the Click-iT EdU buffer additive (component F) by adding 2 mL of deionized water. Mix well and store at -20 &#176;C.</li>
	<li>Store Hoechst 33342 (component G) at 2-6 &#176;C. Store DMSO (component C) in a desiccator at -20 &#176;C. 
	&#8203;NOTE: Gloves should be used to handle DMSO and Hoechst.</li>
</ol>
2. Dissection of third instar larval brains and EdU incorporation
NOTE: The protocol for brain dissections has been described previously12. Before starting dissections, make sure sufficient amounts of the EdU and PFA solutions are prepared and thawed as described in 2.6 and 3.1.
<ol>
	<li>Prepare 10x Phosphate-buffered saline (PBS) by adding 25.6 g Na2HPO4.7H20, 80 g NaCl, 2 g KCl, and 2 g KH2PO4 to 1 L of deionized water. Adjust the pH to 7.4, and subsequently prepare a 1x solution in deionized water.</li>
	<li>Add Schneiders dissection medium (SM) to two consecutive depressions of a glass (3 or 9) depression glass spot plate. Add 1x PBS to the next consecutive depression.</li>
	<li>Using a pair of forceps, pick wandering third instar larvae and place on a tissue wetted with PBS to clean off fly food residue. Place the larva in the depression with PBS and then in the consecutive depression with SM.</li>
	<li>Using a pair of fine forceps, remove 3/4th of the lower body of the larva.
	<ol>
		<li>Gently grab the larval mouth hooks with one pair of forceps, and hold the cuticle at the other end with the other pair of forceps.</li>
		<li>Turn the larval head inside out by pushing inward the mouth hooks and simultaneously peeling off the tissue at the other end.</li>
	</ol>
	</li>
	<li>Observe the larval brain with attached imaginal discs. Remove other tissues attached to the brain, and transfer the brain to the next consecutive depression with SM.</li>
	<li>Thaw the 10 mM EdU stock solution. Prepare a 100 &#956;M solution by diluting this 10 mM stock in SM.</li>
	<li>Add 100 &#956;L of this 100 &#956;M EdU+SM solution in another depression on the Pyrex plate. Incubate ~5-10 dissected brains in 100 &#956;L of 100 &#956;M EdU+SM solution for 2 h at 25 &#176;C. 
	&#8203;NOTE: As NBs divide once in ~2 h, this protocol aims to analyze one full cell cycle. Only use intact brains for further steps. Discard brains that are damaged during dissection.</li>
</ol>
3. Fixation and immunostaining
<ol>
	<li>Prepare 4% paraformaldehyde (PFA) solution in a fume hood.
	<ol>
		<li>Add 4 g of paraformaldehyde powder to 80 mL of 1x PBS in a beaker placed on a magnetic stirrer and heat to 60 &#176;C. To dissolve PFA, add a few drops of 1 N NaOH. Check the pH, and adjust to 7.0 with a few drops of 1 M HCl if required.</li>
		<li>Adjust the volume to 100 mL with 1x PBS. Aliquot the PFA solution and store at 2-6 &#176;C for up to 1 month. Add 0.3% non-ionic detergent (see the Table of Materials) to the PFA solution for efficient fixation of large tissues such as the larval brain.</li>
	</ol>
	</li>
	<li>After EdU incorporation, transfer the brains to 0.6 mL microcentrifuge tubes containing 4% PFA. Incubate at room temperature on a nutator for 15 min. Tap the tube on the laboratory bench so that the brains settle down at the bottom of the tube.</li>
	<li>Remove the PFA and wash 3 times with PBS + 0.3% non-ionic detergent (PBST) at room temperature. Ensure that each wash lasts for at least 10 min on a nutator. After the last wash, remove PBST, add blocking solution (PBST + 5% bovine serum albumin), and incubate for 30 min at room temperature.</li>
	<li>Dilute anti-Miranda antibody (1:250) and anti pH3 antibody (1:200) in PBST (both antibodies in the same tube). Remove the blocking buffer, and add the primary antibody solution. Incubate overnight at 2-6 &#176;C on a nutator. 
	NOTE: Miranda is a membrane protein present on CB NBs. It serves to identify these large round cells in the CB region13.</li>
	<li>Remove the primary antibody solution and wash 3 times with PBST. Ensure that each wash lasts for 10 min on a nutator at room temperature.</li>
	<li>Prepare secondary antibody solution by adding 1:500 diluted Alexa Fluor 488 anti-Rabbit and Alexa Fluor 568 anti-Rat in PBST. Remove the PBST, and add the secondary antibody solution to the dissected brains. Incubate overnight at 2-6 &#176;C in the dark, on a nutator, and wash 3 times with PBST (step 3.5).</li>
</ol>
4. EdU detection, DNA staining, and mounting
<ol>
	<li>&#160;To prepare the EdU detection cocktail, mix the components (see Table 2) in a 1.5 mL tube.</li>
	<li>After the last wash (step 3.6), remove PBST, and add the EdU detection cocktail to the dissected brains. Incubate at room temperature in the dark for 30 min on a nutator. Wash 2 times with PBST for 10 min each, in the dark.</li>
	<li>For DNA staining, dilute Hoechst 33342 (component G) 1:2,000 in PBST to prepare a 5 &#956;g/mL solution.</li>
	<li>Remove PBST after the second wash (step 4.2), and add 500 &#956;L of 5 &#956;g/mL Hoechst solution to the dissected brains. Incubate in the dark for 10 min. Remove Hoechst, and wash once with PBST for 10 min.</li>
	<li>Tap the tube onto the lab bench, and let the brains settle down. Remove the PBST from the tube, but leave behind only 50-100 &#956;L.</li>
	<li>Cut the end of a 200 &#956;L micropipette tip, and carefully transfer the brains onto a clean glass slide. Remove excess PBST from the slide by blotting with filter paper strips. Be careful not to let the filter paper touch the brains.</li>
	<li>Put a drop of 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.</li>
	<li>Gently place a coverslip on top of the brains. Place the slide at 2-6 &#176;C overnight. 
	&#8203;NOTE: The mounting medium hardens upon overnight storage so that there is no need to seal the coverslip with nail polish.</li>
</ol>
5. Imaging
NOTE: See the Table of Materials for details on the laser scanning microscope and oil-immersion objective used in this protocol.
<ol>
	<li>From the Acquisition software, select the 63x objective.</li>
	<li>Put a drop of immersion oil on the coverslip just above the mounted brains to make it easier to locate the tissue through the eyepiece.</li>
	<li>Using the DAPI/Hoechst 33342 channel, find the brain through the eyepiece, and then switch to the acquisition mode in the software.</li>
	<li>Set up 4 channels to image Hoechst 33342 (DNA), Alexa Fluor 488 (pH3), Alexa Fluor 568 (Miranda), and Alexa Fluor 647 (EdU). Use the dye-assistant tool, which automatically sets up the excitation lasers and emission filters for the selected dyes.</li>
	<li>Set the field of view such that it encompasses the entire brain lobe. Image the entire volume of the brain lobe by acquiring z-stacks spaced 0.8 &#956;m apart. Store all images from an imaging session in a *.lif library format.</li>
</ol>
6. Image analysis
NOTE: The following steps describe the analysis of acquired images and how to sort cells into G0/G1 phase, S phase, S&#62;G2/M (progression from S to G2/M), and M phase using the ImageJ software.
<ol>
	<li>Download Fiji (Fiji is ImageJ) from the following URL: https://fiji.sc/. Open Fiji, then drag and drop the .lif files into Fiji. 
	NOTE: Fiji is a version of ImageJ that comes pre-installed with several plugins14. Transferring .lif files into Fiji will open the Bio-formats plugin, which is needed to process the .lif files. Bio-formats plugin is also required to open image files generated from some other microscope brands, e.g., nd2 files generated from a Nikon microscope. This plugin comes pre-installed with Fiji.</li>
	<li>Select Data browser from the stack viewing tab, and use virtual stack from the memory management tab in the bio-formats plugin. 
	NOTE: Lif files with z-stacks from 8-10 brains are often 300-400 megabytes in size. On computers with low RAM, opening a few such files can rapidly deplete the available RAM on ImageJ and prevent any further image processing. Virtual stack is &#39;read-only&#39;, does not need high RAM for processing, and is an ideal option to load large datasets onto Fiji.</li>
	<li>Observe the multichannel image displayed in ImageJ. Change the color of the channels from the menu bar using Image | color | channels tool. Observe the Miranda-labeled NBs as large round cells in the CB region.</li>
	<li>Draw a region of interest (ROI) using the ellipse tool over each NB to avoid counting the NB twice.
	<ol>
		<li>From the ImageJ menu bar, select analyze | tools | ROI manager. Mark all NBs in the current z-section, and press t after marking each cell.</li>
	</ol>
	</li>
	<li>Once all NBs in the current z-section are marked, change the channels to pH3 and EdU, and manually count the number of EdU-positive NBs, pH3-positive NBs, NBs staining positively for both EdU and pH3, and NBs not staining for both markers.</li>
	<li>Search for NBs in subsequent z-sections, delete old ROIs, and add new ROIs to count NBs in different stacks.</li>
	<li>Prepare a spreadsheet with the following columns: 1. EdU- pH3-: this section represents the double-negative cells that are in the G0/G1 phase of the cell cycle; 2. EdU+: the cells in this category have incorporated EdU and are undergoing DNA replication (S-phase); 3. EdU+ pH3+: these dual-positive cells have completed the S phase and have progressed to G2 or M phase; 4. pH3+: these NBs are undergoing mitosis.</li>
	<li>Calculate percentages of NBs present in all of the above 4 categories for each lobe. Prepare a bar graph using the spreadsheet software showing the pooled data for percentages of NBs in each category.</li>
</ol>

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The authors have declared that no competing interests exist.

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) ...

5-ethynyl-2-deoxyuridinephospho-histone-h3-dual-labeling-protocol-for

Research

JoVE Journal

Biology

3.7K Views.  University of Bern. 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. 

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

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

Immunocytochemistry: Human Neural Stem Cells

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of an antigen of interest, most commonly a protein, in cultured cells. This protocol presents an easy-to-follow series of steps that will enable researchers to conserve primary and secondary antibodies while getting high quality, reproducible qualitative and quantitative data out of their staining. There are two aspects of this protocol that help to conserve the volume of antibody necessary for staining. For one, the cells are grown on small, circular coverslips that are placed in wells of a tissue culture plate. After fixation, the cells on coverslips can be removed from the wells of the plate. For antibody staining, the coverslip with cells is inverted onto a small drop of antibody solution on parafilm and is covered with a second piece of parafilm to prevent drying. Using this method, only ~25 &mu;l of antibody solution is needed for each coverslip (or sample) to be stained. This protocol describes immunostaining of human neural stem/precursor cells (hNSPCs), but can be used for many other cell types.

Immunocytochemistry is a powerful method to determine the presence, subcellular localization, and relative abundance of an antigen of interest in cultured cells. This protocol presents an easy-to-follow series of steps that will enable one to conserve antibodies and get the most out of one's staining.

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...