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Monitoring Autophagy in Live Cells Without Transfection

Posted By Brian Conrad
Tags: Autophagy

Transfection is the process of introducing nucleic acids into cells. In cell-based assays, transfection is typically used to investigate protein expression and gene function by introducing nucleic acids (transiently or permanently) into the cell and studying how the cell’s internal machinery modulates gene expression in response. Transfection is most commonly achieved by one of three methods: chemical-based (calcium phosphate, polymers (DEAE-dextran, cationic), activated dendrimer, etc.), cationic lipid-mediated (lipofection) and/or physical (electroporation, cell squeezing, direct microinjection, magnetofection etc.). However, not all methods can be applied to all cell types and experimental circumstances. Transfection efficiencies are dependent upon multiple factors such as the cell type, health of the cells, culture methods being applied, quality and quantity of the DNA being introduced, and the experimental demands. Transfection by way of either chemical-based, lipid-based, or physical methods present a variety of limitations that can affect transfection efficiencies.
Popular chemical-based transfection means such as calcium phosphate co-precipitation are prone to variability; small pH changes can compromise the efficiency of calcium phosphate transfection into the cell and size and quality of the precipitate is critical for successful gene transfer and expression to occur. Likewise, chemical methods used to introduce DNA using DEAE-dextran can become difficult because endocytotic mechanisms that uptake these reagents can accumulate in toxic quantities in the cell. The result is often varying transfection efficiencies which have been found to produce as low as 10% or less successful gene transfer in primary cells. Transfection by way of lipid-mediated methods demonstrates a disadvantage by not having the flexibility to be applied to all cell types unless they have the appropriate frequency of liposomes which is variable across cell types and based on cell health. Lastly, physical methods such as microinjection and electroporation impose different obstacles that both influence transfection efficiency. Microinjections are technically demanding, laborious, and time-consuming. Electroporation does not always lead to high transfection efficiencies especially if studying cells in suboptimal conditions to understand the pathological activities at the molecular level of a disease state.

Autophagy is a dynamic, multi-step stress-induced protective mechanism that can be regulated at several steps. Autophagic activity is typically low under basal conditions, but can be markedly up-regulated by a variety of physiological stimuli such as nutrient starvation, hypoxia, energy depletion, endoplasmic reticulum stress, elevated temperature, high density growth conditions, hormonal stimulation, pharmacological agent treatment, innate immune signaling, and in diseases such as viral, bacterial or parasitic infections as well as various protein aggregopathies (e.g., Alzheimer’s, Huntington’s and Parkinson’s disease), heart disease and acute pancreatitis. The role of autophagic activity in the pathology of cancer, infectious diseases, neurodegeneration, cardiovascular disease, and diabetes has become widely recognized and commonly studied. A reduction in autophagic function is also considered a characteristic of the aging process.

Suboptimal conditions may be imposed on a cell population to study autophagy in a cell-based assay. Cells are subjected to hostile conditions such as nutrient depletion and chemical or environmental stress to induce lysosomal-mediated bulk clearance of cellular debris that autophagic processes utilize. Using transfection-based methods to study such a process in a cell-based assay can often lead to lengthy optimizations and transfection efficiency validation to ensure results are accurate.

To avoid these time consuming, laborious adjustments and validations, Enzo offers a transfection-free CYTO-ID Autophagy Detection Kit and CYTO-ID Autophagy Detection Kit 2.0 for monitoring autophagy in live cells. The kits measure autophagic vacuoles and monitor autophagic flux in lysosomally inhibited live cells using a novel dye that selectively labels accumulated autophagic vacuoles. The dye has been optimized through the identification of titratable functional moieties and allows for minimal staining of lysosomes while exhibiting a bright fluorescent signal once incorporated into pre-autophagosomes, autophagosomes and autolysosomes. Ultimately, this provides the opportunity to study autophagy with a no-transfection quantitative assay which can be utilized on live cells across microscopy, flow cytometry, and microplate platforms. The CYTO-ID Autophagy Detection Kit 2.0 provides a brighter, more photostable dye, which binds to the same acidic, autophagic compartments and can facilitate high-throughput screening of activators and inhibitors of autophagy.

Key Features
  • No transfection required. Eliminates the need for time and effort-consuming transfection efficiency validation required with LC3-fusion protein transfection.
  • Brighter, more photostable edition of our proprietary dye with titratable moieties specific for selectively staining autophagic vesicles.
  • Selective and comprehensive staining that allows for the measurement and differentiation between autophagic flux and autophagolysosome accumulation.
  • Rapidly quantifies autophagy in native heterogeneous cell populations.
  • Validated under a wide range of conditions and with small molecular modulations known to influence autophagy pathways.
  • Optimized protocols for microplate assays, fluorescence microscopy and flow cytometry.
  • Negligible staining of lysosomes reduces background seen with other dyes.
  • Facilitates high-throughput screening of activator and inhibitors of autophagy.

Figure 1. The CYTO-ID® Autophagy Detection Kit specifically labels autophagic vacuoles independent of LC3 protein and eliminates the need for transfection. HeLa cells were subjected to starvation and recovery and then labeled with CYTO-ID® Green detection reagent.

Figure 2. HeLa cells were stained with CYTO-ID® Green Detection Reagent 2 after being cultured in (A) full media or (B) starvation media (EBSS) with 40 µM Chloroquine for 4 hr. Cells starved in EBSS in the presence of Chloroquine showed very bright green fluorescent signals and punctate structures.

Figure 3. Flow cytometry-based profiling of autophagy in Jurkat cells. Jurkat cells were untreated or treated with 0.5 µM Rapamycin (RAP), 10 µM Chloroquine (CLQ) or both for 20 hr. After staining with CYTO-ID® Green Detection Reagent 2 for 30 min, cells were washed and analyzed by flow cytometry. Results are presented as histogram overlay. Cells treated with RAP + CLQ show an increase in fluorescence.

Figure 4. Microplate-based profiling of autophagy in HepG2 cells. HepG2 cells were stained CYTO-ID® Green Detection Reagent 2 after being cultured for 20 hr in DMSO (control), 0.5 µM Rapamycin (Rap), 10 µM Chloroquine (CLQ), or both 0.5 µM Rap and 10 µM CLQ. Cells were also stained with Hoechst 33342 for cell number normalization. Cells treated with both Rapamycin and Chloroquine had an increase in autophagy, as measured by the CYTO-ID® Green Detection Reagent 2.

Figure 5. Eliminate background resulting from non-specific lysosomal staining. CYTO-ID® Green dye eliminates background staining of lysosomes seen with other lysosomotrophic dye-based assays that utilize monodansylcadaverine (MDC) (bottom panel). The CYTO-ID® Autophagy kit eliminates the need for a 350 nm UV laser for live cell analysis, and is compatible for use with Hoechst dyes for co-labeling in microscopy applications.

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Treatment Target Effect μM used Induction Time (hr) Cell Line
Starvation Inhibits mammalian target of Rapamycin (mTOR) Activates autophagy N/A 1~4 HeLa, HepG2, Jurkat
Rapamycin Inhibits mammalian target of Rapamycin (mTOR) Activates autophagy 0.2 6~18 HeLa, Jurkat
PP242 ATP-competitive inhibitor of mTOR Activates autophagy 1 18 HeLa
Lithium Inhibits IMPase and reduce inositol and IP3levels; mTOR-independent Activates autophagy 10,000 18 HeLa, Jurkat
Trehalose Unknown, mTOR - independent Activates autophagy 50,000 6 HeLa, Jurkat
Bafilomycin A1 Inhibits Vacuolar - ATPase Inhibits autophagy 6~9*10-3 18 HeLa, Jurkat
Chloroquine Alkalinizes Lysosomal pH Inhibits autophagy 10~50 18 HeLa, Jurkat
Tamoxifen Increases the intracellular level of ceramide and abolishes the inhibitory effect of PI3K Activates autophagy 4~10 6~18 HeLa, HepG2, Jurkat
Verapamil Ca2+ channel blocker; reduces intracytosolic Ca 2+ levels; mTOR - independent Activates autophagy 40~100 18 HeLa, Jurkat
Hydroxy Chloroquine Alkalinizes Lysosomal pH Inhibits autophagy 10 18 HeLa, Jurkat
Loperamide Ca 2+ channel blocker; reduces intra - cytosolic Ca 2+ levels; mTOR - independent Activates autophagy 5 18 HeLa
Clonidine Imidazoline - 1 receptor agonist; reduces cAMP levels; mTOR - independent Activates autophagy 100 18 HeLa
MG - 132 Selective proteasome inhibitor Activates autophagy 2~5 18 HeLa, Jurkat
Norclomipramine Alkalinizes Lysosomal pH Inhibits autophagy 5 - 20 18 HeLa
Epoxomicin Selective proteasome inhibitor Induce aggresome 0.5 18 HeLa
Velcade ® Selective proteasome inhibitor Induce aggresome 0.5 18 HeLa
Amyloid beta peptide 1 - 42 Induce oxidative stress Induce aggresome 25 18 SK - N - SH

Table 1. Treatments that influence autophagy, previously validated with CYTO-ID® Autophagy Detection Kit (ENZ-51031).

Enzo offers researchers a comprehensive portfolio for the study of autophagy in live cells. Our CYTO-ID® Autophagy Detection Kit 2.0 can be utilized to study autophagosome formation and fusion. In addition, our PROTEOSTAT® Aggresome Detection Kit can detect aggresome/inclusion body formation. We also provide our SCREEN-WELL® Autophagy Detection Library for screening compounds that activate or inhibit autophagic pathways. For further information, please check our Immunoassay & Assay Kits and Compound Libraries platforms or please contact our Technical Support Team for further assistance.

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