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>
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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. 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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 experimental repertoire and reproducibility. These limitations were addressed during the development of our device, which can be used to characterize real time photoswitching kinetics in living cells. To our knowledge, no comparable device exists.
In the current configuration, up to 30 LEDs can be built into one Thermo Flow chamber. Thus, depending on the required light intensities for each wavelength, a single device can be used for a wide variety of optogenetic tools. The established photoswitching protocols can then be optimized for functional readouts, e.g., calcium flux measurements. Our device may be suitable for other optically controlled substances, such as caged compounds or photoactivatable fluorophores.
Depending on the scientific question and specimen used, individualized protocols can be established rapidly. The use of glass tubes instead of polystyrene or polypropylene tubes is recommended to limit light-induced cytotoxicity at wavelengths between 400-500 nm. All tested cell lines survived illumination (360 nm, 400 nm or 500 nm) for up to one hour in glass tubes. We tried to investigate the cause of cell death during illumination in plastic tubes by performing transfer experiments. We illuminated cells in PBS or RPMI with light of different wavelengths and then transferred the supernatant to non-illuminated cells to measure cell death. None of the collected supernatants caused significant cell death in the recipient cells (data not shown). Also, the temperature of illuminated PBS in polystyrene, or polypropylene tubes is almost identical to the temperature in glass tubes. Hence, we can only speculate on the cause of cell death. Illuminated plastic may release an unstable substance or radiation of a wavelength that is toxic for cells.
The choice of medium used for each experiment is important. The buffering capacity must be considered and different reagents, like pH-indicators, absorb different wavelengths to different degrees. Furthermore, cell survival differs significantly, for example, when comparing FCS-free to FCS-containing media.
The goal of light titration is to use the minimal amount of light necessary for maximum photoswitching. For most experiments, it is favorable to maximize the speed of the photoswitch and hence maximize the light intensity. But, depending on the wavelength and cell type, light can have direct effects on the signaling behavior, which is why excessive illumination should be avoided.
It is possible to program light schedules for our device and to connect it to a computer as is common for other light devices. However, since most flow cytometers are in common work places shared by numerous different labs, it is best to maintain the device as small and portable as possible. Furthermore, the manual handling of our device for a two-color setup as presented here is so simple, that programming would only marginally improve the experimental procedure.
Taken together, we present here an innovative device, which combines the power of optogenetic tools and flow cytometry. This will substantially simplify the characterization and development of optogenetic tools in vivo and expand the experimental repertoire.

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><tbody><tr><td>Glass FACS tube</td><td>Thermo Fisher Scientific; Waltham, USA</td><td>14-961-26</td><td>Borosilicate glass tubes 12x75 mm</td></tr><tr><td>Flow Cytometer</td><td>BD Bioscienceg; Heidelberg, Germany</td><td>Fortessa II</td><td>Special Order</td></tr><tr><td>Dronpa: pcDNA3-mNeptune2-N</td><td>Addgene; Cambridge, USA</td><td>41645</td><td></td></tr><tr><td>PolyJet</td><td>SignaGen, Rockville, USA</td><td>SL100688</td><td></td></tr><tr><td>LED 505 nm</td><td>Avago Technologies; Boeblingen, Germany</td><td>HLMP-CE34-Y1CDD</td><td></td></tr><tr><td>LED 400 nm</td><td>Avago Technologies; Boeblingen, Germany</td><td>UV5TZ-400-15</td><td></td></tr><tr><td>Plexiglas tube 15 mm</td><td>Maertin; Freiburg, Germany</td><td>76999</td><td></td></tr><tr><td>Plexiglas 3 mm</td><td>Maertin; Freiburg, Germany</td><td>692230</td><td></td></tr><tr><td>Plexiglas 2.5 mm</td><td>Maertin; Freiburg, Germany</td><td>692225</td><td></td></tr><tr><td>Plexiglas 1.5 mm</td><td>Maertin; Freiburg, Germany</td><td>692215</td><td></td></tr><tr><td>PVC tile 5 mm</td><td>Maertin; Freiburg, Germany</td><td>690020.005</td><td></td></tr><tr><td>PVC tile 6 mm</td><td>Maertin; Freiburg, Germany</td><td>690020.006</td><td></td></tr><tr><td>PVC block 50 mm</td><td>Maertin; Freiburg, Germany</td><td>690020.050</td><td></td></tr><tr><td>RPMI</td><td>Invitrogen, Life Technologies; Darmstadt, Germany</td><td>61870-010</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>EMD; Germany</td><td>805740</td><td></td></tr><tr><td>FCS</td><td>PAN Biotech; Aidenbach, Germany</td><td>P30-3302</td><td></td></tr><tr><td>Penicillin/Strptomycin (10,000 U/mL)</td><td>Invitrogen, Life Technologies; Darmstadt, Germany</td><td>15140-122</td><td></td></tr><tr><td>Acrifix plexiglas glue</td><td>Evonic industries, Essen, Germany</td><td>1R0192</td><td></td></tr><tr><td>Tangit PVC-U glue</td><td>Henkel, D&amp;uuml;sseldorf, Germany</td><td></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 controlled. The center of the device accommodates a standard size FACS tube, which can be connected to most standard flow cytometers (Figures 4 and 5). To show that illumination in our device does not lead to undesired heating of the cells, we measured the temperature of illuminated PBS over time (Figure 6) for three different wavelengths. Illumination with 360 nm, 400 nm or 500 nm leads to only a marginal temperature increase. Overall, the device presented here allows for the reproducible illumination of temperature-controlled cell samples to be measured in a flow cytometer.
Photoswitching of Dronpa
An expression vector encoding a Dronpa-Linker-Dronpa13,16 fusion construct was cloned and transduced into Ramos B cells. As depicted in Figure 7, the goal was to control the conformation of this fusion protein with light of specific wavelengths. Using our device, the cytosolic Dronpa-Linker-Dronpa fusion protein was photoswitched multiple times (Figure 8, left). Switching to the dark state occurs slowly, whereas switching to the bright state happens almost instantaneously. The spontaneous fluorescence recovery of Dronpa in the dark shows that efficient photoswitching to the bright form requires specific illumination and only slowly occurs spontaneously (Figure 8, right).
We performed a kinetic analysis of the photoswitching properties (Figure 9). The slopes for the switching kinetics were calculated for the initial 4 min of illumination. The slope for the spontaneous recovery was calculated for the initial 4 min in the dark.
The kinetic analysis shows, that the Dronpa photoswitching speed is overall very reproducible. In contrast, spontaneous recovery of Dronpa fluorescence in the dark is very inefficient. Even after longer incubation in the dark, only about 10% of the Dronpa fluorescence is recovered. This shows that indeed the illumination in our device induces photoswitching.
Hence, using the described method, we can generate real time photoswitching data in a flow cytometer, which can be translated into a kinetic evaluation of many optogenetic tools.
<img alt="Figure 1" src="/files/ftp_upload/54707/54707fig1.jpg" /> 
Figure 1: Construction plan of the outer layer. All depicted pieces are custom made from polyvinyl chloride (PVC). Piece A: Cylindrical PVC piece with drilled holes to insert LEDs and the tube clips. Piece B: The outer sheath to protect the LEDs from physical damage. Piece C: The tube clip that connects the inner cavity with rubber tubes to allow for water cooling. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54707/54707fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54707/54707fig2.jpg" /> 
Figure 2: Construction plan of the inner layer. All depicted pieces are made from Plexiglas. Piece D: This piece is comprised of three pieces D1, D2 and D3 and forms the unit enclosing the FACS tube. Piece D1: Cylindrical Plexiglas tube. Piece D2: Bottom lid of the inner layer. Piece D3: Lid of the inner layer. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54707/54707fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54707/54707fig3.jpg" /> 
Figure 3: Using the device with a flow cytometer. A1: Sample inlet of a flow cytometer; A2: FACS tube connected to the sample inlet; B: The device enclosing the FACS tube; C: Sample inlet; D: Sample inlet with the device. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54707/54707fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54707/54707fig4.jpg" /> 
Figure 4: Workflow schematic of the device. A: Cells are illuminated in the device (bottom left) and simultaneously taken up and analyzed in the flow cytometer (top left). This allows, for example, the analysis of the kinetic properties of an optogenetic tool (top right). B: cross section of the device. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54707/54707fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54707/54707fig5.jpg" /> 
Figure 5: Process Flow Diagram of the device. One or more LED lights of specific wavelengths illuminate the cell sample in a FACS tube. The LEDs are surrounded by water, which is constantly exchanged by a heated bath circulator to keep the temperature of the cell sample stable. For live cell experiments, the temperature is adjusted to 37 &#176;C. During this temperature-controlled illumination, cells are taken up into the flow cytometer for analysis. This setup allows, for example, to analyze the kinetic properties of an optogenetic tool. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54707/54707fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54707/54707fig6.jpg" /> 
Figure 6: Temperature of illuminated PBS over time. 1 mL of PBS in a glass FACS tube was illuminated over time with 360 nm light, 400 nm light or 500 nm light. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54707/54707fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54707/54707fig7.jpg" /> 
Figure 7: Model for photoswitching of the Dronpa-Linker-Dronpa protein. Illumination with 400 nm light will dimerize (and tetramerize) Dronpa and render it fluorescent. Illumination with 500 nm light will lead to the dissociation of the Dronpa proteins, where monomeric Dronpa is non-fluorescent. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54707/54707fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/54707/54707fig8.jpg" /> 
Figure 8: Characterization of the Dronpa photoswitching properties using the LED Thermo Flow combined with flow cytometry. Cells were illuminated as indicated above the X-axis and the Dronpa mean fluorescence intensity (MFI) is depicted over time. The Dronpa-Linker-Dronpa fusion protein can be photoswitched several times using the device (left). Spontaneous recovery of the Dronpa fluorescence intensity only occurs slowly and inefficiently (right). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54707/54707fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" src="/files/ftp_upload/54707/54707fig9.jpg" /> 
Figure 9: Kinetic analysis of Dronpa photoswitching. The kinetics of Dronpa photoswitching were calculated for the initial 4 min. of illumination and the recovery was calculated for the initial 4 min. incubation in the dark as indicated by the colored areas. The slopes for each calculation are depicted in bold numbers. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54707/54707fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about LED Thermo Flow 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

Universidad San Sebastian

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

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

Genetics

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

Immunology and Infection

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

Bioengineering

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Medicine

Protocol for Co-Culturing Sucrose-Secreting Cyanobacteria with Fungal Partners

Assembly and Quantification of Co-Cultures Combining Heterotrophic Yeast with Phototrophic Sugar-Secreting Cyanobacteria

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being explored as a method for solar-driven bioproduction. One approach involves the use of CO2-fixing cyanobacteria that secrete organic carbon to support the metabolism of a co-cultivated heterotroph, which in turn transforms the carbon into higher-value goods or services. In this protocol, a technical description to assist the experimentalist in the establishment of a co-culture combining a sucrose-secreting cyanobacterial strain with a fungal partner(s), as represented by model yeast species, is provided. The protocol describes the key prerequisites for co-culture establishment: Defining the media composition, monitoring the growth characteristics of individual partners, and the analysis of mixed cultures with multiple species combined in the same growth vessel. Basic laboratory techniques for co-culture monitoring, including microscopy, cell counter, and single-cell flow cytometry, are summarized, and examples of nonproprietary software to use for data analysis of raw flow cytometry standard (FCS) files in line with FAIR (Findable, Accessible, Interoperable, Reusable) principles are provided. Finally, commentary on the bottlenecks and pitfalls frequently encountered when attempting to establish a co-culture with sugar-secreting cyanobacteria and a novel heterotrophic partner is included. This protocol provides a resource for researchers attempting to establish a new pair of co-cultured microbes that includes a cyanobacterium and a heterotrophic microbe.

This protocol provides a comprehensive guideline for the setup and quantitative monitoring of co-cultures, including photoautotrophic sugar-secreting cyanobacteria and heterotrophic yeasts.

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

A Light-Stimulation Grid Approach to Study Cellular Optogenetic Protein

Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid

Our goal was to accurately track the cellular distribution of an optogenetic protein and evaluate its functionality within a specific cytoplasmic location. To achieve this, we co-transfected cells with nuclear-targeted cAMP sensors and our laboratory-developed optogenetic protein, bacterial photoactivatable adenylyl cyclase-nanoluciferase (bPAC-nLuc). bPAC-nLuc, when stimulated with 445 nm light or luciferase substrates, generates adenosine 3&#39;,5&#39;-cyclic monophosphate (cAMP). We employed a solid-state laser illuminator connected to a point scanning system that allowed us to create a grid/matrix pattern of small illuminated spots (~1 &#181;m2) throughout the cytoplasm of HC-1 cells. By doing so, we were able to effectively track the distribution of nuclear-targeted bPAC-nLuc and generate a comprehensive cAMP response map. This map accurately represented the cellular distribution of bPAC-nLuc, and its response to light stimulation varied according to the amount of protein in the illuminated spot. This innovative approach contributes to the expanding toolkit of techniques available for investigating cellular optogenetic proteins. The ability to map its distribution and response with high precision has far-reaching potential and could advance various fields of research.

This protocol involves transfecting cAMP sensors and bPAC-nLuc, an optogenetic protein, to accurately track its cellular distribution and response to light stimulation. The innovative approach of creating a cAMP response map using a point scanning system holds the potential for advancing research with optogenetic proteins in different fields.

LED Thermo Flow — Combining Optogenetics with Flow Cytometry

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