Premo Autophagy Assays with Sensor LC3B, p62, or Tandem Sensor RFP-GFP-LC3B

Easily monitor various stages of cellular autophagy with Premo Autophagy assays and sensors, which are based on LC3B- or p62-linked fluorescent protein chimeras, or the RFP-GFP-LC3B tandem sensor. Premo Autophagy assay kits offer the selectivity of Premo Autophagy sensors, which are based on chimeras of autophagy-associated proteins LC3B or p62, and green (GFP) and/or red fluorescent protein (RFP), with the transduction efficiency of BacMam technology. Transduction or even co-transduction can be performed in a wide variety of cells, including neurons and neural stem cells (NSCs) using a simple protocol that involves adding the BacMam 2.0 chimeric reagent to cells, incubating overnight, and then visualizing transduction using standard GFP (green fluorescent protein) and/or RFP (red fluorescent protein) settings.

Premo Autophagy sensors take advantage of autophagy-associated proteins LC3B or p62, along with GFP or RFP, to detect either the induction or inhibition of autophagy in cells. The Premo Autophagy sensors are transduced using BacMam (insect Baculovirus with a Mammalian promoter) technology, enabling unambiguous visualization of the chimeric proteins in autophagosomes. BacMam reagents are non-replicating in mammalian cells and therefore safe to handle. They are also non-cytotoxic and ready-to-use. Unlike typical expression vectors, BacMam reagents enable titratable and reproducible expression, and offer high co-transduction efficiency; therefore, multiple BacMam reagents can be readily used on the same cells. Recent improvements made to the BacMam system enable efficient transduction in a wider variety of cells, including neurons and neural stem cells (NSCs) with an easy, one-step protocol. To visualize autophagy, simply add the BacMam Autophagy Sensor to cells and incubate overnight for protein expression, then detect signal using standard GFP and/or RFP fluorescence settings on your instrument.

The LC3B protein plays a critical role in autophagy. Normally, this protein resides in the cytosol, but following cleavage and lipidation with phosphatidylethanolamine, LC3B associates with the phagophore. This localization can be used as a general marker for autophagic membranes. The p62 protein, also known as sequestosome (SQSTM1), is an ubitiquitin-binding protein that functions as a receptor for cargos destined to be degraded by the cellular autophagic machinery. When autophagy is induced, the p62 protein localizes to the autophagosomes and is subsequently degraded. Conversely, with the inhibition of autophagy, the p62 protein accumulates in the autophagosome. Thus, the subcellular localization of a p62-fluorescent protein chimera serves as a useful marker for the induction and inhibition of autophagy.

Additionally, by combining the acid-sensitive GFP (i.e., Emerald GFP) with an acid-insensitive RFP (i.e., TagRFP) in the tandem sensor RFP-GFP-LC3B, the change from an autophagosome (neutral pH) to the autolysosome (with an acidic pH) can be visualized by imaging the specific loss of the GFP fluorescence upon acidification of the autophagosome following lysosomal fusion. Upon induction of autophagy, the Premo Tandem Autophagy Sensor labels the punctate autophagosomes; these structures are positive for both GFP and RFP. Once the lysosome has fused, the pH drops, which quenches the GFP, making autolysosomes appear red. Additionally, combining the Premo Autophagy Tandem Sensor with the far-red emitting LysoTracker Deep Red (available separately) allows for a three-color analysis of autophagosomal/autolysomal/lysosomal dynamics.

Each Premo Autophagy assay kit includes chloroquine diphosphate, which is used as a negative control to inhibit autophagy.

Advantages of using Premo Autophagy assays
• Multiplex inspired — combining the Premo Autophagy Tandem Sensor with the far-red emitting LysoTracker Deep Red allows for a three-color analysis of 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 — Premo Autophagy reagents are non-replicating in mammalian cells, lack observable cytopathic effect, and are suitable for biosafety level (BSL) 1 protocols
• Multiple configurations: — Premo Autophagy sensors are available in the following chimeric configurations:
P36235: LC3B-GFP; control LC3B (G120A)-GFP; P36236: LC3B-RFP; control LC3B (G120A)-RFP; P36239: RFP-GFP-LC3B; P36240: GFP-p62; P36241: RFP-p62