LED Thermo Flow - Forening optogenetics med flowcytometri
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 værktøjer har vundet popularitet, dels fordi de kan anvendes til at dechifrere ledningsføring signalveje 1-4. De er baseret på evnen hos fotoaktiverbare proteiner til at ændre deres konformation og bindingsaffinitet, når det belyses med lys. Fusing disse proteiner til signalering elementer giver mulighed for den specifikke regulering af en enkelt spiller i komplekse intracellulære signalveje 5-12. Følgelig kan en signaleringsvej undersøges med høj tidsmæssig og rumlig opløsning.
De fleste cellebaserede optogenetic undersøgelser udnytte mikroskopi-baserede metoder kombineret med dyrkning i nærvær af lys, efterfulgt af biokemisk analyse 11,12. Derimod kan et flowcytometer singularizes celler langs en kapillær og måler cellestørrelse, granularitet og fluorescensintensiteter. Denne metode har store fordele i forhold til mikroskopi eller biokemiske metoder, herunder evnen til at analysere tusinde guldds af levende celler ved enkeltcelle-opløsning i en meget kort tid. Derfor er det ønskeligt at kombinere optogenetics med flowcytometri.
Til vores viden, er der ingen etableret protokol for optogenetic flowcytometri. En bredt accepteret procedure er at manuelt belyse celler udefra reaktionsrøret med lommelygte enheder. Imidlertid manuel belysning i strømmen kræver, cytometer lyset passere gennem reaktionsrøret og for levende celler, en cylindrisk, opvarmet vand kammer. Dette forårsager betydelig lysspredning og lystab. Desuden lysintensiteten billede ved manuel belysningen ikke reproducerbar mellem eksperimenter (vinkel, afstand osv), og der er en praktisk grænse for antallet af bølgelængder i et eksperiment.
Ved at konstruere LED Thermo Flow enhed, var vi i stand til at overvinde disse begrænsninger. Med denne enhed, kan celler belyses med specifikke bølgelængder i en tempratur-styret måde under flowcytometriske målinger. Dette giver mulighed for præcise og reproducerbare mængder af lys i og mellem eksperimenter.
For at demonstrere anvendeligheden af vores enhed in vivo, indspillede vi fluorescens signal om Dronpa i Ramos B-celler under photoswitching. Ramos B-celler afledt af et humant Burkitts lymfom. Dronpa er et fluorescerende protein, der eksisterer som en monomer, dimer eller tetramer. I sin monomere form, det er ikke-fluorescerende. Belysning med 400 nm lys inducerer dimerisation og tetrameriseringsdomæne og gør Dronpa protein fluorescerende. Denne proces kan vendes ved belysning med 500 nm lys. Den Dronpa proteinet er blevet anvendt til at styre funktionen og placeringen af signalproteiner 4,13.
Her udtrykte vi en Dronpa-linker-Dronpa protein i Ramos B-celler til at studere photoswitching af Dronpa i et flowcytometer. Ved hjælp af vores enhed, var vi i stand til effektivt ogreproducerbart photoswitch Dronpa mens du optager sin fluorescensintensitet i realtid. Denne metode giver betydelige fordele i forhold til de nuværende belysning protokoller med manuel belysning og betydeligt udvider den eksperimentelle repertoire for optogenetic værktøjer og bur forbindelser. Ved hjælp af vores enhed i væsentlig grad vil forenkle og fremskynde opdagelse og udvikling af nye optogenetic værktøjer.
Protocol
Representative Results
Discussion
LED Thermo Flow er en innovativ enhed til at studere optogenetic værktøjer i et flowcytometer.
Hidtil har optogenetic prøver blevet belyst kun med mikroskopi lasere eller lommelygte enheder 11,12. Afhængig af vinklen og afstanden af lommelygten til prøven forventes betydelig variation i mængden af belysning mellem eksperimenter. Desuden er der en grænse for antallet af lommelygter en enkelt person kan operere i et eksperiment. Dette begrænser den eksperimentell…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
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 (10000 U/ml) | Invitrogen, Life Technologies; Darmstadt, Germany | 15140-122 | |
| Acrifix plexiglas glue | Evonic industries, Essen, Germany | 1R0192 | |
| Tangit PVC-U glue | Henkel, Düsseldorf, Germany |
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