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
MilliporeSigma’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
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).
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)
| Description | Catalogue 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 |