We thank J. Schmidt from the University of Freiburg for constructing the device. We thank P. Nielsen and D. Medgyesi for their support and critical reading of this manuscript. This study was supported by the Deutsche Forschungsgemeinschaft through SFB746 and by the Excellence Initiative of the German Research Foundation (GSC-4, Spemann Graduate School).

1. Designing and Building the Device
<ol>
	<li>Pilot Experiments 
	NOTE: The required light intensity for a specific optogenetic tool and cell type may vary significantly. Pilot experiments with prototypes are useful to estimate the minimal light intensity necessary. The optogenetic tool used for the following experiments is a Dronpa-Linker-Dronpa fusion construct. The Dronpa sequence was commercially obtained (See List of Materials). A long linker was cloned in between two Dronpa sequences to allow the expressed construct to form intramolecular (and intermolecular) dimers. The steps described below can be adapted to many other optogenetic tools or optically controlled substances.
	<ol>
		<li>Transduce Ramos B lymphocytes with a Dronpa-Linker-Dronpa containing construct following the manufacturer&#39;s instructions for transduction using the retroviral-containing supernatant from packaging cells. Harvest and count cells according to a previously published protocol13.</li>
		<li>Resuspend cells at a concentration of 1 x 106/mL in medium (RPMI 1640, 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin, and 50 &#181;M 2-mercaptoethanol) and transfer them to a glass FACS tube.</li>
		<li>Insert the tube into a flow cytometer and measure the fluorescence intensity of Dronpa (GFP channel)14,15.</li>
		<li>Wear protective glasses for all further steps to protect eyes from possibly damaging wavelengths.</li>
		<li>Manually place a 500 nm LED light on the outside of the tube and continue measuring the fluorescence intensity of Dronpa.</li>
		<li>Increase the number and/or intensity of the LED lights until Dronpa fluorescence intensity decreases.</li>
		<li>Manually place a 400 nm LED light on the outside of the tube and continue measuring the fluorescence intensity of Dronpa.</li>
		<li>Increase the number and/or intensity of the LED lights until the Dronpa fluorescence intensity increases.</li>
		<li>Use at least as many LED lights for building the device as predicted to be necessary from the photoswitching in steps 1.1.5 and 1.1.7.</li>
	</ol>
	</li>
	<li>Building the device 
	NOTE: The device was built by a professional machining shop, the Arbeitsgruppe Technik from the University of Freiburg. Most parts are custom made and not commercially available. The exact dimensions of each part are depicted in Figures 1 and 2. To clarify the assembly of this device, Supplementary Video 1 is provided and the most essential steps are described below.
	<ol>
		<li>Mill piece A from polyvinyl chloride (PVC) with the exact measurements shown in Figure 1A.</li>
		<li>Insert LED lights into the drilled holes of piece A and seal each hole with PVC glue to prevent water leakage.</li>
		<li>Connect LEDs to a multichannel transformer (each wavelength should occupy a separate channel) with an adjustable output power from 0-29 mA.</li>
		<li>Attach the outer sheath (B) with 3 screws to piece A as depicted to protect the LEDs from physical damage.</li>
		<li>To be able to connect the device to a water pump, glue in the tube clips (C) into the drilled holes of piece A (depicted in Figure 1a, cross-section Y: 9 mm and 61.5 mm) using PVC glue.</li>
		<li>Cut a 58.5 mm long Plexiglas tube (D1).</li>
		<li>Manufacture pieces D2 and D3 from Plexiglas as depicted in Figure 2.</li>
		<li>Glue piece D3 to the bottom of D1 with Plexiglas glue.</li>
		<li>Attach a rubber O-ring to piece D2 and glue it to the top of D1 with Plexiglas glue. 
		NOTE: The pieces D1, D2 and D3 together form piece D.</li>
		<li>Insert piece D into piece A with 3 screws.</li>
		<li>Test the device for water leakage prior to applying power to the transformer.</li>
	</ol>
	</li>
</ol>
2. Measuring the Kinetics of an Optogenetic Tool
<ol>
	<li>Prepare cells
	<ol>
		<li>Culture Ramos B cells in RPMI-medium (RPMI 1640, 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin, and 50 &#181;M 2-mercaptoethanol) at a density of 3-10 x 106 cells/mL at 37 &#176;C and 5% CO2.</li>
		<li>Harvest Ramos B cells by centrifugation at 350 x g and 4 &#176;C for 5 min.</li>
		<li>Count cells using a Neubauer counting chamber following the manufacturer&#39;s instructions and resuspend 1-5 x 106 cells in 600 &#181;L medium.</li>
		<li>Transfer the cells to a glass FACS tube, put them on ice and protect them from light until the measurement.</li>
	</ol>
	</li>
	<li>Prepare the flow cytometer and the device.
	<ol>
		<li>Connect the device to a heated water pump and adjust the temperature to 37 &#176;C.</li>
		<li>When using glass FACS tubes, exchange the black O-ring of the flow cytometer with a rubber O-ring and additionally apply silicon grease.</li>
	</ol>
	</li>
	<li>Measurement
	<ol>
		<li>Carefully insert the glass FACS tube into the device and connect it to the flow cytometer (Figure 3).</li>
		<li>Start the measurement and record Dronpa fluorescent intensity (GFP channel).</li>
		<li>Illuminate the cell sample with 400 nm or 500 nm light respectively and record the Dronpa fluorescence intensity (GFP channel).</li>
	</ol>
	</li>
	<li>Data Analysis
	<ol>
		<li>Analyze experimental data with suitable flow cytometer software. Plot forward scatter against sideward scatter and gate the living cells. Plot the Dronpa fluorescence intensity over time.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Dugue, G. P., Akemann, W., Knopfel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+concept+of+optogenetics.">A comprehensive concept of optogenetics.</a> Prog Brain Res. 196, 1-28 (2012).</li><li>Tischer, D., Weiner, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+cell+signalling+with+optogenetic+tools.">Illuminating cell signalling with optogenetic tools.</a> Nat Rev Mol Cell Biol. 15 (8), 551-558 (2014).</li><li>Toettcher, J. E., Voigt, C. A., Weiner, O. D., Lim, W. 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+promise+of+optogenetics+in+cell+biology:+interrogating+molecular+circuits+in+space+and+time.">The promise of optogenetics in cell biology: interrogating molecular circuits in space and time.</a> Nat Methods. 8 (1), 35-38 (2011).</li><li>Zhang, K., Cui, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+control+of+intracellular+signaling+pathways.">Optogenetic control of intracellular signaling pathways.</a> Trends Biotechnol. 33 (2), 92-100 (2015).</li><li>Bugaj, L. J., Choksi, A. T., Mesuda, C. K., Kane, R. S., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+protein+clustering+and+signaling+activation+in+mammalian+cells.">Optogenetic protein clustering and signaling activation in mammalian cells.</a> Nat Methods. 10 (3), 249-252 (2013).</li><li>Bugaj, L. 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=Regulation+of+endogenous+transmembrane+receptors+through+optogenetic+Cry2+clustering.">Regulation of endogenous transmembrane receptors through optogenetic Cry2 clustering.</a> Nat Commun. 6, 6898 (2015).</li><li>Strickland, 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=TULIPs:+tunable,+light-controlled+interacting+protein+tags+for+cell+biology.">TULIPs: tunable, light-controlled interacting protein tags for cell biology.</a> Nat Methods. 9 (4), 379-384 (2012).</li><li>Taslimi, 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=An+optimized+optogenetic+clustering+tool+for+probing+protein+interaction+and+function.">An optimized optogenetic clustering tool for probing protein interaction and function.</a> Nat Commun. 5, 4925 (2014).</li><li>Toettcher, J. E., Gong, D., Lim, W. A., Weiner, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-based+feedback+for+controlling+intracellular+signaling+dynamics.">Light-based feedback for controlling intracellular signaling dynamics.</a> Nat Methods. 8 (10), 837-839 (2011).</li><li>Toettcher, J. E., Weiner, O. D., Lim, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+optogenetics+to+interrogate+the+dynamic+control+of+signal+transmission+by+the+Ras/Erk+module.">Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module.</a> Cell. 155 (6), 1422-1434 (2013).</li><li>Wang, 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=Utilization+of+a+photoactivatable+antigen+system+to+examine+B-cell+probing+termination+and+the+B-cell+receptor+sorting+mechanisms+during+B-cell+activation.">Utilization of a photoactivatable antigen system to examine B-cell probing termination and the B-cell receptor sorting mechanisms during B-cell activation.</a> Proc Natl Acad Sci U S A. 113 (5), E558-E567 (2016).</li><li>Wend, 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=Optogenetic+control+of+protein+kinase+activity+in+mammalian+cells.">Optogenetic control of protein kinase activity in mammalian cells.</a> ACS Synth Biol. 3 (5), 280-285 (2014).</li><li>Zhou, X. X., Chung, H. K., Lam, A. J., Lin, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+control+of+protein+activity+by+fluorescent+protein+domains.">Optical control of protein activity by fluorescent protein domains.</a> Science. 338 (6108), 810-814 (2012).</li><li>Baumgarth, N., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+approach+to+multicolor+flow+cytometry+for+immunophenotyping.">A practical approach to multicolor flow cytometry for immunophenotyping.</a> J Immunol Methods. 243 (1-2), 77-97 (2000).</li><li>Givan, A. 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The authors declare that they have no competing financial interests.

The LED Thermo Flow is an innovative device to study optogenetic tools in a flow cytometer.
So far, optogenetic samples have been illuminated only with microscopy lasers or flashlight devices11,12. Depending on the angle and distance of the flashlight to the sample, substantial variability in the amount of illumination is expected between experiments. Furthermore, there is a limit to the number of flashlights a single person can operate in an experiment. This restricts the experimen...

Optogenetic tools have been gaining popularity, in part because they can be used to decipher the wiring of signaling pathways1-4. They are based on the ability of photoactivatable proteins to change their conformation and binding affinity when illuminated with light. Fusing these proteins to signaling elements allows for the specific regulation of a single player within complex intracellular signaling pathways5-12. Consequently, a signaling pathway can be studied with high temporal and spatial resolution.
Most cell-based optogenetic studies utilize microscopy-based methods combined with culturing in the presence of light, followed by biochemical analysis11,12. In contrast, a flow cytometer singularizes cells along a capillary and measures cell size, granularity and fluorescence intensities. This method has major advantages over microscopy or biochemical methods, including the ability to analyze thousands of living cells at single-cell resolution in a very short time. Hence, it is desirable to combine optogenetics with flow cytometry.
To our knowledge, there is no established protocol for optogenetic flow cytometry. A broadly accepted procedure is to manually illuminate cells from outside the reaction tube with flashlight devices. However, manual illumination in the flow cytometer requires the light to pass through the reaction tube and, for live cell imaging, a cylindrical, heated water chamber. This causes substantial light scattering and loss of light. Moreover, the light intensity provided by manual illumination is not reproducible between experiments (angle, distance, etc.) and there is a practical limit to the number of wavelengths in one experiment.
By constructing the LED Thermo Flow device, we were able to overcome these limitations. With this device, cells can be illuminated with specific wavelengths in a temperature-controlled manner during flow cytometric measurements. This allows for precise and reproducible amounts of light within and between experiments.
To demonstrate the utility of our device in vivo, we recorded the fluorescence signal of Dronpa in Ramos B cells during photoswitching. Ramos B cells are derived from a human Burkitt&#39;s lymphoma. Dronpa is a fluorescent protein that exists as a monomer, dimer or tetramer. In its monomeric form, it is non-fluorescent. Illumination with 400 nm light induces dimerization and tetramerization and renders the Dronpa protein fluorescent. This process can be reversed by illumination with 500 nm light. The Dronpa protein has been used to control the function and location of signaling proteins4,13.
Here, we expressed a Dronpa-Linker-Dronpa protein in Ramos B cells to study photoswitching of Dronpa in a flow cytometer. Using our device, we were able to efficiently and reproducibly photoswitch Dronpa while recording its fluorescence intensity in real time. This method provides substantial advantages over current illumination protocols with manual illumination and significantly broadens the experimental repertoire for optogenetic tools and cage compounds. Using our device will significantly simplify and accelerate the discovery and development of novel optogenetic tools.

<table border="0" cellpadding="0" cellspacing="0" width="1212">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Glass FACS tube</td>
			<td style="width:357px;">&#160;Thermo Fisher Scientific; Waltham, USA</td>
			<td style="width:312px;">14-961-26</td>
			<td style="width:296px;">Borosilicate glass tubes 12x75 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Flow Cytometer</td>
			<td style="width:357px;">BD Bioscienceg; Heidelberg, Germany</td>
			<td style="width:312px;">Fortessa II</td>
			<td>Special Order</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Dronpa: pcDNA3-mNeptune2-N</td>
			<td style="width:357px;">Addgene; Cambridge, USA</td>
			<td style="width:312px;">41645</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PolyJet</td>
			<td style="width:357px;">SignaGen, Rockville, USA</td>
			<td style="width:312px;">SL100688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">LED 505 nm</td>
			<td style="width:357px;">Avago Technologies; Boeblingen, Germany</td>
			<td style="width:312px;">HLMP-CE34-Y1CDD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">LED 400 nm</td>
			<td style="width:357px;">Avago Technologies; Boeblingen, Germany</td>
			<td style="width:312px;">UV5TZ-400-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Plexiglas tube 15 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">76999</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Plexiglas 3 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">692230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Plexiglas 2,5 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">692225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Plexiglas 1,5 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">692215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PVC tile 5 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">690020.005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PVC tile 6 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">690020.006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PVC block 50 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">690020.050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">RPMI</td>
			<td style="width:357px;">Invitrogen, Life Technologies; Darmstadt, Germany</td>
			<td style="width:312px;">61870-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">2-Mercaptoethanol</td>
			<td style="width:357px;">EMD; Germany</td>
			<td style="width:312px;">805740</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">FCS</td>
			<td style="width:357px;">PAN Biotech; Aidenbach, Germany</td>
			<td style="width:312px;">P30-3302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Penicillin/Strptomycin (10000 U/ml)</td>
			<td style="width:357px;">Invitrogen, Life Technologies; Darmstadt, Germany</td>
			<td style="width:312px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Acrifix plexiglas glue</td>
			<td style="width:357px;">Evonic industries, Essen, Germany</td>
			<td style="width:312px;">1R0192</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Tangit PVC-U glue</td>
			<td style="width:357px;">Henkel, D&#252;sseldorf, Germany</td>
			<td style="width:312px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

led thermo flow &#8212; combining optogenetics with flow cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

Using the LED Thermo Flow with a Flow Cytometer
The functional core of the device is a cylindrical chamber in which LED lights are arranged in a circular manner pointing inward. This chamber can be connected to a water supply and pump, which allows for controlling the temperature of the LEDs, as well as the cell sample. The LEDs are connected to a transformer and hence the light intensity for each wavelength can be individually co...

Watch this Scientific Journal Video about LED Thermo Flow Ã¢â‚¬â€ Combining Optogenetics with Flow Cytometry at JoVE.com

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

led-thermo-flow-combining-optogenetics-with-flow-cytometry

Research

JoVE Journal

Biology

8.5K Views.  Max-Planck Institute for Immunobiology und Epigenetics. Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

Video: LED Thermo Flow — Combining Optogenetics with Flow Cytometry

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

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 ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...