Premo autophagy tandem sensor rfp gfp lc3b kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit is a fluorescence-based assay that enables real-time monitoring of autophagy flux in live cells. The kit contains a plasmid encoding a fusion protein of red fluorescent protein (RFP), green fluorescent protein (GFP), and the autophagy marker LC3B. This tandem sensor reports on the different stages of autophagy through the relative fluorescence of GFP and RFP signals.

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37 protocols using premo autophagy tandem sensor rfp gfp lc3b kit

Autophagy Assessment by Tandem RFP-GFP-LC3B Assay

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The Premo Autophagy Tandem Sensor RFP‐GFP‐LC3B kit (P36239; Life Technologies, Carlsbad, CA) was used as the assay for cellular autophagic flux. The LC3B is tagged with both GFP and RFP, thereby enabling differentiation of autophagosomes from autolysosomes, as acid‐sensitive GFP is degraded by lysosomal enzymes once fusion between autophagosomes and lysosomes occurs and the autolysosomal environment becomes acidic. Therefore, autophagosomes are shown as yellow puncta‐positive cells, whereas autolysosomes show only red puncta. The cell preparation for confocal microscopic exams was performed. The number of cells positive for yellow or red puncta was counted after examining 50 cells in 10 random fields (5 cells/field) under a × 40 confocal microscope objective. Confocal microscopic images were obtained using the ZEN software on a Zeiss 780 with the following filters: Ex/Em 488/510 nm for GFP and Ex/Em 568/603 nm for RFP. All experiments were performed in at least triplicate per condition.

Ha Y., Fang Y., Romecin Duran P.A., Tolosa E.J., Moser C.D., Fernandez‐Zapico M.E, & Roberts L.R. (2019). Induction of Lysosome‐associated Protein Transmembrane 4 Beta via Sulfatase 2 Enhances Autophagic Flux in Liver Cancer Cells. Hepatology Communications, 3(11), 1520-1543.

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Autophagy Monitoring in MDA-MB-231 Cells

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MDA-MB-231 cells (1 × 10⁴ cells/well) were plated in a Nunc™ 177,437 Lab-Tek Chamber Slide System (Thermo Fisher Scientific, Rochester, NY, USA) and allowed to adhere overnight. The various stages of autophagy were monitored by the Premo™ Autophagy Tandem Sensor RFP-GFP LC3B Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions, which achieved efficient transduction using insect Baculovirus carrying the acid-sensitive LC3B-fluorescent protein chimera with a mammalian promoter. After the cells were treated overnight with 3 μL of BacMam reagents containing RFP-GFP-LC3B DNA, specified concentrations of drugs were subsequently added to the cell medium for 48 h. Aloperine (100 μM) was the positive control and induced autophagic flux, which was blocked by CQ. The media were removed, and live cell imaging solution containing Hoechst 33342 (1 µg/mL) was added and incubated for 20 min in the dark. The cells were then washed with 1× phosphate-buffered saline (PBS), covered in mounting medium (F4680) (Sigma-Aldrich, Inc. St. Louis, MO, USA), and imaged using a Zeiss laser scanning confocal microscope LSM800 (Carl Zeiss Microscopy GmbH, Jena, Germany).

Chen M.S., Yeh H.T., Li Y.Z., Lin W.C., Lee Y.R., Tseng Y.S, & Sheu S.M. (2020). Flavopereirine Inhibits Autophagy via the AKT/p38 MAPK Signaling Pathway in MDA-MB-231 Cells. International Journal of Molecular Sciences, 21(15), 5362.

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Autophagy Flux in H/R-Injured NRK-52E Cells

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Normoxia-cultured and H/R-injured NRK-52E cells were co-cultured with HMSCs in the presence or absence of 3-MA. Autophagic flux was measured by a Premo Autophagy Tandem Sensor RFP-GFP-LC3B kit (Life Technologies, Rockville, MD) according to the manufacturer’s instructions. Fluorescence images were acquired after 48 h of incubation and imaged using a ×63 objective on a confocal fluorescence microscope (FV10i, Olympus). The LC3B puncta of ten non-overlapping images were quantified using the Image J software.

Tseng W.C., Lee P.Y., Tsai M.T., Chang F.P., Chen N.J., Chien C.T., Hung S.C, & Tarng D.C. (2021). Hypoxic mesenchymal stem cells ameliorate acute kidney ischemia-reperfusion injury via enhancing renal tubular autophagy. Stem Cell Research & Therapy, 12, 367.

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Autophagy Modulation in Prostate Cancer

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PC-3 and DU 145 cells were plated at a density of 4.0 x 10⁴ cells/well in 6-well plate, and left to adhere overnight in a 37°C incubator with 5% CO₂. All cells were transduced with RFP-GFP-LC3B reagent using commercially available Premo Autophagy Tandem Sensor RFP-GFP-LC3B Kit (Life Technologies, USA) for 48 h. The following day, cells were incubated with 100 μM chloroquine diphosphate (CQ), and treated with either DMDP-1, DMDP-2 or left untreated (control) for 24 h. The cells were visualized under an inverted fluorescent microscope (Nikon Instruments, Japan) using a blue filter to detect the accumulation of GFP-LC3 punctate with GFP emission.

Suparji N.S., Chan G., Sapili H., Arshad N.M., In L.L., Awang K, & Hasima Nagoor N. (2016). Geranylated 4-Phenylcoumarins Exhibit Anticancer Effects against Human Prostate Cancer Cells through Caspase-Independent Mechanism. PLoS ONE, 11(3), e0151472.

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Tracking Autophagy Dynamics in Breast Cells

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To analyse NA-induced autophagosome and autolysosome formation over time in MCF-7, MDA-MB-231, and MCF-12A cells, we used the commercially available Premo Autophagy Tandem Sensor RFP-GFP-LC3B Kit (Life technologies, Carlsbad, CA, USA). All three cell lines were plated in 35-mm dishes and allowed to reach 70% confluence by culturing for 24 h before NA treatment. All cells were simultaneously transduced with the RFP-GFP-LC3B reagent and treated with NA for 48 h. The cells were visualised with a live cell-imaging microscope using standard green fluorescent protein (GFP) and red fluorescent protein (RFP) settings. The neutral pH autophagosome, identified by GFP emission, and acidic autolysosome, identified by RFP emission, were visualised every 1 h for up to 48 h.

Suman S., Das T.P., Reddy R., Nyakeriga A.M., Luevano J.E., Konwar D., Pahari P, & Damodaran C. (2014). The pro-apoptotic role of autophagy in breast cancer. British Journal of Cancer, 111(2), 309-317.

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Autophagy Visualization in Cells

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Cells were seeded on glass coverslips and cultured in CM and DM for 72 h. The day after seeding, the cells were transfected with Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions, and fixed in PBS containing 4% paraformaldehyde and 2% sucrose, pH 7.6. The principle of this assay resides in the fact that the low pH in the lysosomes quenches the fluorescence of GFP, whereas the fluorescence of RFP remains stable. Upon the formation of autophagosomes the number of GFP-positive/RFP-positive (yellow) vesicles is increased. These vesicles become GFP-negative/RFP-positive (red) after fusion with lysosomes.
Finally, the coverslips were mounted using ProLongTM Gold antifade reagent (Thermo Fisher Scientific, Waltham, MA, USA). Images were acquired with a SP8 Leica confocal microscope using a 40× objective in oil.

Zocchi M., Locatelli L., Zuccotti G.V., Mazur A., Béchet D., Maier J.A, & Castiglioni S. (2022). Magnesium Homeostasis in Myogenic Differentiation—A Focus on the Regulation of TRPM7, MagT1 and SLC41A1 Transporters. International Journal of Molecular Sciences, 23(3), 1658.

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Visualizing Lipid Droplet Dynamics and Autophagy

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To explore intracellular localization of lipid droplets with subcellular organelles, after differentiation with rosiglitazone or EPA, lipid droplets were co-stained with BODIPY 493/503 and ER-Tracker or LysoTracker following the manufacture’s (Molecular Probes) instruction. To monitor autophagic flux, hMGECs were transduced with the Premo Autophagy Tandem Sensor RFP-GFP-LC3B kit (Molecular Probes, Cat# P36239) and lipid droplets were stained with Lipi-blue (Dojindo Molecular Technologies, Rockville, MD). Briefly, hMGECs were seeded at 40,000 cells onto 35 mm glass bottom culture dishes, grown to 70% confluence and transduced with LC3B reagent followed by 48 h of incubation before imaging. This tandem RFP-GFP sensor capitalizes on the pH difference between the acidic autolysosome and the neutral autophagosome and the pH sensitivity differences exhibited by GFP (green fluorescent protein) and RFP (red fluorescent protein) to monitor progression from the autophagosome (both green and red) to autolysosome (red). Intracellular green and red dots were counted in 50 cells and an average count per cell was analyzed for relative fraction of autolysosome for analysis of autophagic flux.

Kim S.W., Rho C.R., Kim J., Xie Y., Prince R.C., Mustafa K., Potma E.O., Brown D.J, & Jester J.V. (2020). Eicosapentaenoic acid (EPA) activates PPARγ signaling leading to cell cycle exit, lipid accumulation, and autophagy in human meibomian gland epithelial cells (hMGEC). The ocular surface, 18(3), 427-437.

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Autophagy Evaluation in CTEPH PAECs

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The PAECs collected from CTEPH rats were transfected with mRFP-GFP-LC3 plasmids for 24 h using the Premo^™ Autophagy Tandem Sensor RFP-GFP-LC3B kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. Next, the PAECs were imaged by fluorescence microscopy (×20 magnification; Hitachi High-Technologies Corporation) and quantified using ImageJ software (24 ).

Wu Q., Zhou X., Wang Y, & Hu Y. (2022). LncRNA GAS5 promotes spermidine-induced autophagy through the miRNA-31-5p/NAT8L axis in pulmonary artery endothelial cells of patients with CTEPH. Molecular Medicine Reports, 26(4), 297.

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Autophagic Flux Analysis in Huh7 Cells

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For GFP-LC3 and lamp1 colocalization, Huh7 cells were transfected with GFP-LC3 plasmid for 48 h and cells were then seeded to a cover glass slide chamber and treated with WSG, HBSS (Gibco) or Baf A1 (Selleck). Immunofluorescence staining was performed as previously described [57 (link)].
For autophagic flux analysis, Huh7 cells were seeded and grown overnight on a 20-mm-glass-bottom cell-culture dish (NEST, 801001). The formation of autolysosomes in cells treated with WSG and CQ was detected using the Premo Autophagy Tandem Sensor RFP-GFP-LC3B Kit (Thermo Fisher Scientific, P36239) according to the manufacturer's protocol. Nuclei were stained in FBS-free DMEM medium contained 5 ng/ml hoechst (Thermo Fisher scientific) at 37 °C for 10 min.
For GFP-LC3 puncta assay, cells were transfected with GFP-LC3 plasmid for 48 h, and then plated on a 20-mm-glass-bottom cell-culture dish and treated with WSG. All fluorescent images were taken using confocal microscopy (Carl Zeiss Meditec, Inc.) and quantified using Image J software.

Wang N., Liu H., Liu G., Li M., He X., Yin C., Tu Q., Shen X., Bai W., Wang Q., Tao Y, & Yin H. (2020). Yeast β-D-glucan exerts antitumour activity in liver cancer through impairing autophagy and lysosomal function, promoting reactive oxygen species production and apoptosis. Redox Biology, 32, 101495.

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Autophagic Flux Monitoring in Cells

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The Premo Autophagy Tandem Sensor RFP-GFP-LC3B Kit (P36239, Thermo Fisher Scientific, Waltham, MA) was used to assess autophagic flux in the patient dermal fibroblasts and BMSCs. Briefly, 1 × 10⁴ cells were plated in each well of a 24-well plate and cultured overnight. Cells were then transduced with the BacMam 2.0 RFP-GFP-LC3B reagent at 40 particles per cell for 24 h. The autophagy inhibitor chloroquine (50 μM for 4 h, Thermo Fisher Scientific, Waltham, MA) was used as a positive control for the generation of autophagosomes and the autophagy inducer rapamycin (1 μM for 2 h, 37094, Sigma-Aldrich, St. Louis, MO) was used as a positive control for the generation of both autophagosomes and autolysosomes. After viral transduction, the cells were fixed in 4% paraformaldehyde for 15 min, stained with Hoechst 33342, and mounted with ProLong Gold Antifade Mountant (Thermo Fisher Scientific, Waltham, MA) and allowed to cure for 24 h prior to imaging. Cells were imaged using a Zeiss LSM 700 Confocal Microscope coupled with a C-Apochromat 40×/1.20 W Korr M27 objective. Images were processed using the ZEN software and analyzed using ImageJ.

Li C., Brazill J.M., Liu S., Bello C., Zhu Y., Morimoto M., Cascio L., Pauly R., Diaz-Perez Z., Malicdan M.C., Wang H., Boccuto L., Schwartz C.E., Gahl W.A., Boerkoel C.F, & Zhai R.G. (2017). Spermine synthase deficiency causes lysosomal dysfunction and oxidative stress in models of Snyder-Robinson syndrome. Nature Communications, 8, 1257.

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