The Premo Autophagy Tandem Sensor RFP-GFP-LC3B Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Do not freeze component A	- Incubate the cells overnight (≥16 hours) after adding LC3 reagent. - For best results, allow cells 48 hours post-transduction for expression levels to equilibrate

Upstream tips
- Do not freeze component A
Protocol tips
- Incubate the cells overnight (≥16 hours) after adding LC3 reagent. - For best results, allow cells 48 hours post-transduction for expression levels to equilibrate

Publication protocol

The formation of autophagosome and autophagolysome in A375 cells in control and NS 1643 treated cells was detected using the Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit (Thermo Fischer Scientific, Grand Island, NY, USA) as described in the manufacturer's instructions. The RFP-GFP-LC3B sensor enables the detection of LC3B positive, neutral pH autophagosomes in green fluorescence (GFP) and LC3B positive acidic pH autophagolysosme in red fluorescence (RFP). The cells were grown on coverslips and incubated with 6 μL of BacMam Reagents containing the RFP-GFP-LC3B overnight. The cells were then treated with either vehicle alone, NS 1643 at 50 μM concentration, or hydroxychloroquine sulfate (CalBiochem) for 16 hours. The cells were rinsed in 1X DPBS, nuclei were stained with DAPI The coverslips were mounted with VECTASHIELD reagent (Vector Laboratories, Burlingame, CA) and fluorescent images were taken using confocal microscopy (Carl Zeiss Meditec, Inc., Thornwood, NY). LC3B positive autophagosomes (green) and LC3B positive autophagolysosome (red) were analyzed and quantified using Image J.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for The Premo Autophagy Tandem Sensor RFP-GFP-LC3B Kit below.

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