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.

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.

优点：

数据：

Premo™ Autophagy Sensor Tandem LC3B can be used to discriminate between acidic and neutral LC3B-positive vesicles.

HeLa cells were transduced with 40 particles per cell of Premo™ Autophagy Tandem Sensor and cultured for 24 hours. Cells were then incubated in either Vehicle, 90uM Chloroquine or 200uM Leupeptin A for a further 24 hours. Cells were imaged on a Zeiss LSM confocal microscope using standard filter sets for GFP and RFP. Chloroquine blocks autophagy through neutralization of Lysosomal pH and hence fluorescence is seen from both GFP and RFP. Leupeptin A blocks autophagy without changing lysosomal pH hence GFP fluorescence is lost in the acidic environment of the autolysosome whereas RFP retains fluorescence.

Schematic depiction of the autophagy pathway in a eukaryotic cell.

The first step involves the formation and elongation of the isolation membranes or phagophore. The second step entails the expansion and sequestering of the cytoplasm and formation of the double-membrane autophagosome and includes the association of the cytosolic LC3 protein. Fusion of lysosome with autophagosome to generate the autolysosome is the penultimate step. In the fourth and final phase, the cargo is degraded.

The use of the Premoâ„¢ Autophagy Tandem Sensor RFP-GFP-LC3B allows for 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.

相关产品：

▪Premo™ Autophagy Sensor RFP-p62 Kit（货号#P36241）

▪Premo™ Autophagy Sensor GFP-p62 Kit（货号#P36240）

▪Premo™ Autophagy Sensor GFP-LC3B Kit（货号#P36235）

▪Premo™ Autophagy Sensor RFP-LC3B Kit（货号#P36236）

▪Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit（货号#P36239）