Actiflash - A photoinducible protein activator

Actiflash has been successfully used with various cell lines as well as animal models:
● Cell lines: CV1, mammalian epithelial cells (MDCK)
● Zebrafish: 12 hpf – 2 dpf embryos
● Mice

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Actiflash works optimally on embryos below 2 dpf when  following the provided protocol of use. On older embryos, protocol adjustments might need to be implemented to achieve a satisfactory photoconversion (i.e. heart injection, electroporation, etc).   

You will find below a non-exhaustive list of proteins that have been fused to the estrogen receptor:
● Recombinase Cre
● Tyrosine-protein kinase ABL1
● Cellular tumor antigen p53
● Proto-oncogene c-Fos
● Endothelial transcription factor GATA-2
● CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase4 (GAL4)

Actiflash is provided as a powder. It is stable for years at 2-8°C. Once solubilized in a DMSO solution (typically at 10 mM), Actiflash can be stored at -20°C for several months.  Always wrap the vials containing the aliquoted Actiflash with aluminium foil to protect it from light. 

You can perform experiments with Tamoxifen-OH as a positive control.

After conditioning your sample at a given concentration (e.g. 5 μM) with Actiflash,
illuminate your samples with the shortest illumination duration leading to the maximum
phenotype recovery. Compare with the results obtained with Tamoxifen-OH (1 μM).

Any plasmid containing the ERT sequence should work. 

Please find below a list of 52 plasmids developed by Sophie Vriz and Michel Volovitch in The Sophie Vriz Lab, who have the ownership of citations related to these plasmids. These 52 plasmids are all available on Addgene.

With the Actiflash conditioned samples, analyze the phenotype recovery observed using a
100% laser power and a decreasing illumination duration. You will thus determine the
shortest illumination duration leading to 100% uncaging.

Examples of the duration required for uncaging 100% of Actiflash:

• One-photon excitation at a 365 nm wavelength with a benchtop UV lamp having a power
range of 4-6 W (e.g. for global photoactivation): 5 minutes.
• One-photon excitation in the 350-405 nm, laser excitation power P = 10 μW focused on a
spot of diameter 10 μm: a few seconds.
• Two-photon excitation delivering 200 fs pulses at 750 nm and P = 10 mW in a cell of volume
V =100–1000 μm3: 1 second.