For autophagy detection, 1×103 cardiomyocytes were seeded into 96-well plates. Cultured cardiomyocytes were transduced with the adenovirus GFP-LC3 (Invitrogen, USA) at a multiplicity of infection of 60 for 24 h. The number of GFP-LC3 dots was determined by manual counting in 5 fields, and nuclear number was evaluated by counting DAPI-stained nuclei in the same fields using the same magnification. The number of GFP-LC3 puncta/cell was evaluated as the total number of dots divided by the number of nuclei in each microscopic field.
Gfp lc3
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GFP-LC3 is a fluorescent protein-based reporter for monitoring autophagy in cells. It consists of the green fluorescent protein (GFP) fused to the autophagy-related protein LC3. This reporter allows for the visualization and quantification of autophagic structures within cells.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
1640 medium, by Thermo Fisher Scientific (1 mentions)
Cell culture consumables, by Corning (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
C1 vector, by Addgene (1 mentions)
Hsp70 shrna, by Santa Cruz Biotechnology (1 mentions)
Gfp lc3, by Addgene (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
5 protocols using gfp lc3
1
Quantifying Autophagy in Cardiomyocytes
2
Cell Culture Conditions for U2OS and MCA205 Cell Lines
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U2OS cell lines were cultured in Dulbecco’s modified Eagle’s medium (#41 966-02, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (#F7524, Sigma Aldrich), 1% non-essential amino acids (#11 140-035, Thermo Fisher Scientific), 1% HEPES (#15630080, Thermo Fisher Scientific) and 1% penicillin/streptomycin (#15140122, Thermo Fisher Scientific). For U2OS HMGB1-GFP, XBP1ΔDBD-venus-RFP-FYVE, GFP-LC3 and GFP-ATF6 0.5 mg/mL G418 (#10 131-27, Thermo Fisher Scientific) was added to the medium. MCA205 cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (#61870044, Thermo Fisher Scientific) supplemented with identical components. Cells were maintained in a humidified incubator at 37°C with 5% CO2. Cell culture consumables were purchased from Corning (New York, USA).
3
Visualizing Autophagy in SH-SY5Y Cells
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GFP-LC3 was firstly transfected into SH-SY5Y cells. After transfection 48 h, the cells were followed by a cycle of washing with PBS and detected fluorescence intensity via a fluorescence microscope purchased from Olympus Corporation (USA). The GFP-LC3 was purchased from Thermo Fisher (USA) [13 (link)].
4
Plasmid Transfection and Knockdown in Cell Lines
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GFP-LC3 was obtained from Addgene (Cambridge, MA, USA) (Plasmid 21073). Full-length parkin cDNA was subcloned into a C1 vector (Addgene plasmid 54607) to generate a GFP-tagged parkin construct. GFP-LC3 and GFP-parkin were transfected into HEK293T and HeLa cells, respectively, using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Negative control shRNA (shNC), parkin shRNA, Hsp70 shRNA and Bim shRNA were purchased from Santa Cruz Biotechnology. The viral particles were combined with 8 μg/mL polybrene and used to infect HEK293T cells overnight. The cell culture medium was replaced with fresh complete growth medium, and after 48 h, the cells were used for experiments.
5
GFP-LC3 Puncta Analysis in K562/ADM Cells
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K562/ADM cells were transfected with GFP-LC3 (Invitrogen) for 48 h by using lipofectamine 3000 (Invitrogen). Then these cells were treated with MAT (0, 0.5, 1, 2 mg/mL) for 48 h. After washed with PBS, the LC3+ punctate fluorescence was observed under a fluorescence microscope.
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