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 all...

<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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+an+introduction.">Flow cytometry: an introduction.</a> Methods Mol Biol. 699, 1-29 (2011).</li><li>Brakemann, T., 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=A+reversibly+photoswitchable+GFP-like+protein+with+fluorescence+excitation+decoupled+from+switching.">A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching.</a> Nat Biotechnol. 29 (10), 942-947 (2011).</li></ol>

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 â€” Combining Optogenetics with Flow Cytometry at JoVE.com

LED Thermo Flow &amp;#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.

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 ...

The overall goal of this method is to stimulate optically controlled substances during flow cytometric measurements. This novel device can help answer key questions in the field of cell signaling by combining the power of optically controlled substances with flow cytometry. For the first time, optogenetic tools can be characterized and analyzed using flow cytometry.No comparable established method existed until today. To begin, suspend transduced Ramos B lymphocytes at a concentration of one million cells per milliliter and transfer them to a glass FACS tube. Next, exchange the black O-ring of the flow cytometer with a rubber O-ring and apply silicone grease to create a better seal.Insert the glass tube into a flow cytometer and place the LED prototype around the tube with the lights off. Measure the fluorescence intensity of Dronpa in the GFP channel. The most critical step of the procedure is handling of the glass tubes.Glass FACS tubes are very fragile and, hence, it is recommended to practice the insertion of the glass tubes into the cytometer prior to doing experiments. Then, switch on the 500 nanometer LED light on the outside of the tube, and continue measuring the flourescence intensity of Dronpa. Increase the intensity of the LED lights until Dronpa fluorescence intensity decreases, in order to estimate the minimal light intensity needed, in order to measure the photoswitching of the Dronpa-Linker-Dronpa protein.Next, switch on the 400 nanometer LED light of the prototype at the outside of the tube, and continue measuring the fluorescence intensity of Dronpa. Increase light intensity of the 400 nanometer LED lights until Dronpa fluorescence intensity increases. Start by following the design and manufacture instructions in the accompanying text protocol to fabricate the inner and outer layers of the photoswitching LED device.Insert the 400 and 500 nanometer wavelength LEDs into the channels of the device. Glue them into place and wire them as directed. These specific wavelengths will be used to switch the Dronpa-Linker-Dronpa fusion proteins between their dark and bright states.Then, assemble the device and make sure it is water-tight in order to prevent leakage prior to powering up the transformer. Following device assembly, harvest Ramos B lymphocytes and suspend them in a density of three to ten million cells per milliliter. Transfer 600 microliters of the cells to a glass FACS tube.Place them on ice and protect the cells from light until they are measured. Connect the assembled device to a heated water pump and adjust the temperature to 37 degrees Celsius. Then, carefully insert the glass FACS tube into the device and connect it to the flow cytometer.Record the Dronpa fluorescent intensity in the GFP channel. The design of this optogenetic devices includes a chamber that can be connected to a circulating water supply to maintain the temperature of sample at a constant value. While the device emitted three different wavelengths, only a marginal temperature increase was seen in the FACS sample, when flowing 37 degree Celsius water through the device.The Dronpa-Linker-Dronpa fusion construct was cloned and transduced into Ramos B cells. When directed into the monomer state by a 500 nanometer light source, the emission intensity decreases. Then, once the light source is switched to the 400 nanometer light, the Dronpa monomer is quickly changed into the dimer state, and the intensity quickly increases.Using this device, the cytosolic Dronpa-Linker-Dronpa fusion protein was able to be photoswitched multiple times. 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.The kinetic analysis shows that the photoswitching speed is overall very reproducible and that even after a long incubation in the dark, only about ten percent of the fluorescence is recovered without photoswitching. Following this procedure, any flow cytometric analysis, including calcium flux measurements, can be performed in order to explore the functionality of your chosen optogenetic tool. After it's development, this technique will pave the way for researchers in the field of optogenetics to explore the wiring of cell signaling pathways.After watching this video, you should have a good understanding of how to establish a photoswitching protocol for your specific optogenetic tool. Don't forget that UV light can be extremely hazardous and precaution, such as protective glasses, should always be worn while performing this procedure.

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

Research

JoVE Journal

Biology

8.4K Aufrufe.  Max-Planck Institute for Immunobiology und Epigenetics. The overall goal of this method is to stimulate optically controlled substances during flow cytometric measurements. This novel device can help answer key questions in the field of cell signaling by combining the power of optically controlled substances with flow cytometry. For the first time, optogenetic tools can be characterized and analyzed using flow cytometry.No comparable established method existed until today. To begin, suspend transduced Ramos B lymphocytes at a concentration ...