What is it for? Use it if you want to subtlely control a protein expression from a spatial and/or temporal point of view. Or if you want to perform in vivo cell tracking experiments. Use it as well if you want to easily switch on or off with light your CRE-ERT2 model.
Ludovic Jullien, Isabelle Aujard and Thomas Le Saux
Reviewed by David Bensimon, Lorenzo Bombardelli, Sidney Cambridge, Cristina Pujades, Angel Raya, Alexandre Specht, Perrine de Villemagne
Caged Cyclofen-OH is cell-permeant and can be added either in the external medium or directly injected for conditioning.
Excellent chemical stability
Caged Cyclofen-OH does not generate any basal activation of protein function and it benefits from an excellent temporal resolution upon uncaging.
Favorable wavelength ranges for uncaging
Uncaging requires either UV-A light or a strong IR laser. Visible light is inactive, which facilitates the experiments with biological samples.
Caged Cyclofen-OH liberates Cyclofen-OH, which is photostable in contrast to Tamoxifen-OH encountering photodegradation under illumination.
Wide applicative scope
Technology capitalizing on the versatile use of Tamoxifen-OH for controlling functions of multiple types of proteins.
Actiflash in powder is stable for years at 2-8°C, protected from light
Actiflash in solution (no water) has to be stored at -20°C, protected from light
Actiflash is provided as a powder and is usually dissolved in spectrophotometric grade DMSO to get 10 mM solutions
DMSO solutions should be aliquoted in Eppendorfs (one Eppendorf should be used for one or two experiments).
#1 Unfreeze only the Eppendorf you will use for your daily experiment
#2 Before use, homogeneize the solution with a pipette to be sure that it will be homogenous (crystallization may occur upon DMSO freezing)
#3 Carefully calibrate the photoactivation of Actiflash (concentration for conditioning, power of the light source, geometry and duration of illumination). Then always work under the same conditions.
It is advised to first establish the extent of phenotype sought for as a function of the Tamoxifen-OH concentration. Then the concentration of Actiflash used for sample conditioning is fixed at Tamoxifen-OH concentration causing 100% of the desired phenotype (in general 3-5 μM in cultured cells and zebrafish embryos).
The caged precursor Actiflash has to absorb photons to liberate Cyclofen-OH. The effective photoactivation wavelengths are comprised between 325 and 425 nm for one-photon excitation, which makes benchtop UV lamp or light source installed on microscopes relevant light sources. Two different methods are proposed for fixing the illumination features for the Actiflash uncaging.
. Method 1
In the first method, uncaging is directly evidenced by analyzing in the Actiflash-conditioned sample the dependence of the phenotype recovery on the illumination duration (e.g. a few min with a benchtop UV lamp at 365 nm at a few cm from the sample surface).
. Method 2
In the second method, the light source needs is calibrated for the photon flux it generates at the biological sample. To proceed, one can use either a power-meter. Once the light source is calibrated, one can calculate the duration of illumination to fully liberate Actiflash from its caged precursor.
Once established, the Actiflash concentration for sample conditioning, power of the light source, and geometry and duration of illumination must remain constant to get reproducible results.
Actiflash and us
"Once upon a time, a physicist (David Bensimon) asked a chemist (Ludovic Jullien) whether he could design a caged inducer to photocontrol protein activity in living organisms. For sure! However we also needed a biologist (Sophie Vriz) to accept the challenge to validate the caged Cyclofen-OH technology. It has been a long but so nice adventure, which has involved the tight integration of the work from many talented students, postdocs, and collaborators... Thanks to all of them!"
Contact the team about their technology
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