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Summary

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.

Abstract

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.

Introduction

Optogenetic tools have been gaining popularity, in part because they can be used to decipher the wiring of signaling pathways^1-4. 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 pathways^5-12. 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 analysis^11,12. 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'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 proteins^4,13.

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.

Protocol

1. Designing and Building the Device

1. 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.
   1. Transduce Ramos B lymphocytes with a Dronpa-Linker-Dronpa containing construct following the manufacturer's instructions for transduction using the retroviral-containing supernatant from packaging cells. Harvest and count cells according to a previously published protocol¹³.
   2. 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 µM 2-mercaptoethanol) and transfer them to a glass FACS tube.
   3. Insert the tube into a flow cytometer and measure the fluorescence intensity of Dronpa (GFP channel)^14,15.
   4. Wear protective glasses for all further steps to protect eyes from possibly damaging wavelengths.
   5. Manually place a 500 nm LED light on the outside of the tube and continue measuring the fluorescence intensity of Dronpa.
   6. Increase the number and/or intensity of the LED lights until Dronpa fluorescence intensity decreases.
   7. Manually place a 400 nm LED light on the outside of the tube and continue measuring the fluorescence intensity of Dronpa.
   8. Increase the number and/or intensity of the LED lights until the Dronpa fluorescence intensity increases.
   9. 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.
2. 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.
   1. Mill piece A from polyvinyl chloride (PVC) with the exact measurements shown in Figure 1A.
   2. Insert LED lights into the drilled holes of piece A and seal each hole with PVC glue to prevent water leakage.
   3. Connect LEDs to a multichannel transformer (each wavelength should occupy a separate channel) with an adjustable output power from 0-29 mA.
   4. Attach the outer sheath (B) with 3 screws to piece A as depicted to protect the LEDs from physical damage.
   5. 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.
   6. Cut a 58.5 mm long Plexiglas tube (D1).
   7. Manufacture pieces D2 and D3 from Plexiglas as depicted in Figure 2.
   8. Glue piece D3 to the bottom of D1 with Plexiglas glue.
   9. 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.
   10. Insert piece D into piece A with 3 screws.
   11. Test the device for water leakage prior to applying power to the transformer.

2. Measuring the Kinetics of an Optogenetic Tool

1. Prepare cells
   1. 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 µM 2-mercaptoethanol) at a density of 3-10 x 10⁶ cells/mL at 37 °C and 5% CO₂.
   2. Harvest Ramos B cells by centrifugation at 350 x g and 4 °C for 5 min.
   3. Count cells using a Neubauer counting chamber following the manufacturer's instructions and resuspend 1-5 x 10⁶ cells in 600 µL medium.
   4. Transfer the cells to a glass FACS tube, put them on ice and protect them from light until the measurement.
2. Prepare the flow cytometer and the device.
   1. Connect the device to a heated water pump and adjust the temperature to 37 °C.
   2. When using glass FACS tubes, exchange the black O-ring of the flow cytometer with a rubber O-ring and additionally apply silicon grease.
3. Measurement
   1. Carefully insert the glass FACS tube into the device and connect it to the flow cytometer (Figure 3).
   2. Start the measurement and record Dronpa fluorescent intensity (GFP channel).
   3. Illuminate the cell sample with 400 nm or 500 nm light respectively and record the Dronpa fluorescence intensity (GFP channel).
4. Data Analysis
   1. 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.

Representative Results

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…

Discussion

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 devices^11,12. 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.

Materials

Glass FACS tube	Thermo Fisher Scientific; Waltham, USA	14-961-26	Borosilicate glass tubes 12×75 mm
Flow Cytometer	BD Bioscienceg; Heidelberg, Germany	Fortessa II	Special Order
Dronpa: pcDNA3-mNeptune2-N	Addgene; Cambridge, USA	41645
PolyJet	SignaGen, Rockville, USA	SL100688
LED 505 nm	Avago Technologies; Boeblingen, Germany	HLMP-CE34-Y1CDD
LED 400 nm	Avago Technologies; Boeblingen, Germany	UV5TZ-400-15
Plexiglas tube 15 mm	Maertin; Freiburg, Germany	76999
Plexiglas 3 mm	Maertin; Freiburg, Germany	692230
Plexiglas 2.5 mm	Maertin; Freiburg, Germany	692225
Plexiglas 1.5 mm	Maertin; Freiburg, Germany	692215
PVC tile 5 mm	Maertin; Freiburg, Germany	690020.005
PVC tile 6 mm	Maertin; Freiburg, Germany	690020.006
PVC block 50 mm	Maertin; Freiburg, Germany	690020.050
RPMI	Invitrogen, Life Technologies; Darmstadt, Germany	61870-010
2-Mercaptoethanol	EMD; Germany	805740
FCS	PAN Biotech; Aidenbach, Germany	P30-3302
Penicillin/Strptomycin (10,000 U/mL)	Invitrogen, Life Technologies; Darmstadt, Germany	15140-122
Acrifix plexiglas glue	Evonic industries, Essen, Germany	1R0192
Tangit PVC-U glue	Henkel, Düsseldorf, Germany