The Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit combines the ability to monitor the various stages of autophagy (through LC3B protein localization) with the high transduction efficiency and minimal toxicity of BacMam 2.0 expression technology. To perform image-based analysis for autophagy, simply add the BacMam 2.0 RFP-GFP-LC3B reagent to your mammalian cells, incubate overnight to ensure maximum protein expression, and then visualize using standard GFP (green fluorescent protein) and RFP (red fluorescent protein) settings.
This tandem RFP-GFP sensor capitalizes on the pH difference between the acidic autolysosome and the neutral autophagosome and the pH sensitivity differences exhibited by GFP (green fluorescent protein) and RFP (red fluorescent protein) to monitor progression from the autophagosome to autolysosome. The RFP and the GFP genes included in this chimera are TagRFP and Emerald GFP, respectively.
Each Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit includes BacMam 2.0 RFP-GFP-LC3B reagent and chloroquine diphosphate, which is used to inhibit autophagy.
• Multiplex inspired: combining the Premo™ Autophagy Tandem Sensor with the far-red emitting LysoTracker® Deep Red (available separately) allows for a three-color analysis of the complete autophagic pathway dynamics
• Highly efficient delivery system: >90% transduction of a wide range of mammalian cell lines, including primary cells, stem cells, and neurons
• Fast and convenient: simply add the Premo™ Autophagy reagent to your cells, incubate overnight, and image—or store frozen, assay-ready cells for later use
• Robust: the Premo™ Autophagy reagents are non-replicating in mammalian cells, lack observable cytopathic effect, and are suitable for biosafety level (BSL) 1
The LC3B protein plays a critical role in autophagy, and the localization of this protein to autophagosomes can be used as a general marker for formation of autophagosomes during stimulation of autophagy and/or the accumulation of autophagosomes during inhibition of autophagic flux.
Currently available non-tandem autophagy chimeras, such as Premo™ Autophagy LC3B-GFP (P36235) and LC3B-RFP (P36236), are useful for monitoring autophagosome formation, especially when combined with other chimeras or fluorescent reagents. However, the Premo™ Autophagy Tandem Sensor allows an enhanced dissection of the maturation of the autophagosome to the autolysosome. By combining an acid-sensitive GFP with an acid-insensitive RFP, the change from autophagosome (neutral pH) to autolysosome (with an acidic pH) can be visualized by imaging the specific loss of the GFP fluorescence, leaving only red fluorescence. In addition, combining the Premo™ Autophagy Tandem Sensor with the far-red emitting LysoTracker® Deep Red (L12492) allows for a three-color analysis of the autophagosomal/autolysosomal/lysosomal dynamics.
To ensure reliable, high levels of expression in a wide variety of mammalian cells, the Premo™ Autophagy Sensors utilize BacMam 2.0 expression technology. BacMam reagents (insect Baculovirus with a Mammalian promoter) do not replicate in mammalian cells, are non-cytoxic (biosafety level 1), and are ready to use. Unlike expression vectors, BacMam reagents enable titratable and reproducible expression and offer high co-transduction efficiency; therefore, multiple BacMam reagents can be used in the same cell.
Each Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit includes a vial of chloroquine diphosphate. Chloroquine has been demonstrated to inhibit autophagy by elevating lysosomal pH and therefore inhibiting the fusion of autophagosomes with lysosomes and preventing the subsequent lysosomal protein degradation.