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GFP- & RFP-LC3 Analysis by Flow Cytometry

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LentiBrite™ LC3-II Enrichment Kit (Catalogue No. 17-10230)

GFP- & RFP-LC3 Analysis of Autophagy by Flow Cytometry
Analysis of GFP-LC3 localization in HUVEC by flow cytometry.
Analysis of GFP-LC3 localization in HUVEC by flow cytometry. HUVECs were lentivirally transduced with TagGFP2-LC3 wild-type (GFP-LC3 wt, top row) or TagGFP2-LC3G120A control mutant (GFP-LC3 mutant, center row). U2OS cells stably expressing TagGFP2-LC3 wild-type were also analyzed (U2OS-GFP-LC3, bottom row). Transduced cells were detached and either permeabilized to release free, cytosolic LC3 (green peaks) or left intact (gray peaks). After processing, the cells were analyzed by flow cytometry on a guava easyCyte™ 8HT instrument. Upon permeabilization, only TagGFP2-LC3 wild-type-expressing cells under starvation conditions display retention of the fusion protein, indicative of tight association of LC3 with autophagosomes. (Click image to enlarge.)
To more accurately assess the extent of LC3-II localization to the autophagosome in primary cells, flow cytometry assays can be employed. However, the presence of cytosolic LC3-I (non-lipidated form) can cause persistent background signal and can make quantitative analysis difficult via flow cytometry.

Merck’s LentiBrite LC3-II Enrichment Kit for flow cytometry employs a proprietary technique to selectively permeabilized the plasma membrane, such that free cytosolic fluorescent protein-tagged LC3 is released while autophagosome-bound LC3 fusion protein is retained.

HUVECs were lentivirally transduced with TagGFP2-LC3 or TagGFP2-LC3G120A (control mutant). The cells were subsequently starved of amino acids in the presence of a lysosome inhibitor or left untreated, then detached and either permeabilized (using the LentiBrite Autophagosome Enrichment Kit) or left intact.

Upon permeabilization, the GFP-LC3 in starved cells was almost completely retained, but was greatly depleted in fed cells. In contrast, permeabilization caused a large reduction in TagGFP2-LC3G120A in both starved and fed cells. In U2OS cells transiently transfected with plasmid encoding TagGFP2-LC3, a very broad distribution of fluorescence was observed, and the shift upon permeabilization of fed cells was much less pronounced (data not shown).

FlowCellect™ GFP-LC3 Reporter Cell Line Autophagy Assay Kits

Flow cytometry detection of LC3 translocation to autophagosomes by selective permeabilization.
Flow cytometry detection of LC3 translocation to autophagosomes by selective permeabilization The FlowCellect™ GFP-LC3 Reporter Autophagy Kit was used without selective permeabilization to show high levels of GFP-LC3 within the cytoplasm before and after starvation (induction of autophagy) in (A).

Following selective permeabilization, GFP-LC3 level remains high in autophagosomes when starved and in the presence of lysosome inhibitor (green); even without the inhibitor, a slight shift is observed when starved (blue) as shown in (B). All cytosolic GFP-LC3 is washed away if no autophagy is induced by starvation (gray).
Merck’s optimized FlowCellect™ kits take the guesswork out of assay development so you can focus on your research. Our newest kits provide a quantitative solution for studying autophagy and measuring the potency of autophagy inducers using flow cytometry. Choose from formats specific for flow cytometry (FCCD100170) or both flow cytometry and imaging (FCCH100181).

Like all FlowCellect™ assays, these are validated for easyCyte™ benchtop flow cytometers as well as other flow cytometry instruments.

FlowCellect™ Autophagy Kit Advantages:

  • Sensitive - Included selective permeabilization solution enables measurement of LC3-GFP translocation by extracting cytosolic (but not autophagosome-sequestered) LC3-GFP
  • Accurate - Monomeric GFP reporter minimizes dimer formation and aggregation, facilitating translocation and flow cytometry analysis.
  • Robust - Since autophagy is a constitutive cellular degradation process, the use of lysosomal inhibitors prevents the degradation of GFP-LC3, prolonging the signal and enabling quantification by flow cytometry.
  • Low cost – stable cell lines allow repeated use of the assays, in combination with the Autophagy Detection Reagent Pack (CF200097)

DescriptionCatalogue No.
FlowCellect™ LC3-GFP Reporter Autophagy Assay Kit (CHO) FCCH100170
FlowCellect™ LC3-GFP Reporter Autophagy Assay Kit (U2OS) FCCH100181
FlowCellect™ Autophagy Detection Reagent Pack, 100 tests CF200097