Cells were transfected with the stubRFP-sensGFP-LC3 lentivirus purchased from GENE-CHEM following the manufacturer’s protocol. After transfection with mRFP-GFP-LC3, autophagosomes were labeled yellow (mRFP and GFP) whereas autolysosomes were labeled red (mRFP only). Then cells were visualized using fluorescence microscopy.
Stubrfp sensgfp lc3 lentivirus
Manufactured by Genechem
Sourced in China
The StubRFP-sensGFP-LC3 lentivirus is a gene delivery tool used for fluorescence-based monitoring of cellular processes. It expresses both a red fluorescent protein (RFP) and a green fluorescent protein (GFP) fused to the autophagy marker LC3. This lentiviral system can be used to study autophagy-related dynamics in live cells.
Automatically generated - may contain errors
11 protocols using stubrfp sensgfp lc3 lentivirus
1
Visualizing Autophagy Dynamics via Fluorescence
2
Stable Cell Line Generation for Autophagy Assay
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The stub-RFP-sens-GFP-LC3 lentivirus was obtained from GeneChem Co. Ltd (Shanghai, China). To construct stable cell lines expressing stub-RFP-sens-GFP-LC3, Capan-1 and SW1990 cells plated in 96-well plates were cultured overnight and then infected with lentivirus. The multiplicity of infection was 20 for Capan-1 and SW1990 cells. Infected cells were selected by 3 μg/mL puromycin (Solarbio Life Science, Beijing, China). When GFP-RFP-LC3 autophagic puncta were detected, cells were cultured on 24-well cell cover glasses, fixed with 4% paraformaldehyde for 15 min, and stained with 1 μg/mL diamidino-2-phenyl indole for 10 min. The staining process was performed at room temperature and cells were protected from light. The GFP-RFP-LC3 autophagic puncta were examined using a Leica SP5 confocal microscope (Leica Microsystems Inc., Germany) with a 63× oil immersion lens.
3
Visualizing Autophagy with Fluorescent Markers
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Cells were transfected with the stubRFP-sensGFP-LC3 lentivirus purchased from GENE-CHEM following the manufacturer’s protocol. Then, cells were visualized using laser scanning confocal microscopy (LSM800, Zeiss). Autophagosomes were labeled yellow (mRFP and GFP) whereas autolysosomes were labeled red (mRFP only).
4
Autophagic Flux Monitoring in Chondrocytes
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The stubRFP-sensGFP-LC3 lentivirus were constructed by Genechem Co (Shanghai, China). The primary passage 1 chondrocytes were seeded in 6-well plates, and after overnight culture were transduced with the lentivirus in serum-free medium at a multiplicity of infection (MOI) of 50. Complete α-MEM was used to replace media 12 h post infection, and the cells was grown for 24 h further while being treated with IL-1β, PD and 3-MA. The autophagic flux was observed using a laser scanning confocal microscope (Nikon America Inc., Melville, NY), and the stubRFP and sensGFP punctae were counted manually in at least 40 cells per sample.
5
Assessing Autophagic Flux in NPCs
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The stubRFP-sensGFP-LC3 Lentivirus (GeneChem, China) was used to assess autophagic flux. NPCs were spread onto 24-well plates at a density of 1 × 105 cells/well 24 hours prior to lentivirus transfection. The density of NPCs at the time of lentiviral transfection was set at approximately 2 × 105 cells/well. The next day, the medium was replaced with 2 mL of fresh medium containing 7 µg/mL polybrene, and 2 µl of virus suspension was added. After 24 hours, the medium containing the virus was replaced with fresh medium, and incubation was continued. The cells were observed daily under a fluorescence microscope. After the indicated treatment, the cells were obtained under an inverted fluorescence microscope.
6
Stable Cell Line for Autophagy Monitoring
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We obtained the stubRFP-sensGFP-LC3 lentivirus from GeneChem Corporation (Shanghai, China). After being cultivated to a confluence of about 20% in 24-well plates, BRL3A cells were co-incubated with stubRFP-sensGFPLC3 lentivirus for 48 h. Then, the positive cells were chosen using 4 g/mL puromycin (Solarbio, Beijing, China) to create stable cell lines. The GFP-RFP-LC3-marked cells were seeded on glass coverslips in 24-well plates. The complete medium was replaced with serum-free media at 80% confluence, and the cells were then treated in accordance with the guidelines of the study. Then, the fluorescence images of LC3 puncta were viewed using a fluorescence microscope (TCS SP8 STED, Leica). The yellow LC3 puncta indicate autophagosomes, and the red LC3 puncta indicates autolysosomes.
7
Visualizing Autophagy with mRFP-GFP-LC3
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The cells were transfected with stubRFP-sensGFP-LC3 lentivirus, which was obtained from GENE-CHEM and the manufacturer's protocol was followed. Following transfection with mRFPGFP-LC3, yellow labeling (mRFP and GFP) and red labeling (mRFP only) were used to label autophagosomes and autolysosomes, respectively. Finally, fluorescence microscopy was used to visualize the cells.
8
Monitoring Autophagy Flux using StubRFP-SensGFP-LC3
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StubRFP-sensGFP-LC3 lentivirus were purchased from Genechem (Shanghai, China) and were used to monitor autophagic flux. The MDA-MB-231 and MDA-MB-468 cell lines were infected with StubRFP-sensGFP-LC3 lentivirus, and after stable expression, they were transfected with sh-con and sh-Linc00707 plasmids. Furthermore, to further verify whether Linc00707 affects autophagy through the PI3K/AKT/mTOR signaling pathway, The PI3K inhibitor LY294002 10 μmol/L was added to the above cells. The StubRFP-SensGFP-LC3 fluorescence spots were photographed under laser confocal microscopy (TCS SP8, Leica, Germany), to monitor the progress of intracellular autophagic flux.
9
Visualizing Autophagic Flux with mRFP-GFP-LC3
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Cells were transfected with the stubRFP-sensGFP-LC3 lentivirus purchased from GENE-CHEM following the manufacturer's protocol. After transfection with mRFP-GFP-LC3, autophagosomes were labelled yellow (mRFP and GFP) whereas autolysosomes were labelled red (mRFP only). Then cells were visualized using uorescence microscopy.
10
Monitoring Autophagy Flux with stubRFP-sensGFP-LC3
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We purchased tandem stubRFP-sensGFP-LC3 lentiviruses (Genechem Company, Shanghai, China) to monitor autophagy flux. Well-conditioned cells were seeded into 6-well plates and transfected with stubRFP-sensGFP-LC3 lentiviruses for 24 hours. Cells stably expressing stubRFP-sensGFP-LC3 were selected using puromycin (5 ug/ml) treatment. After the corresponding intervention for 2 days, cells were observed using a Nikon Eclipse Ti-U fluorescence microscope.
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