HPCs were cultured on confocal dishes for 5 days and grown to 40–50% confluence at the time of transfection. After being washed with PBS two times, the cells were transfected with Ad-mCherry-GFP-LC3B adenovirus (Beyotime, C3011, China) at a multiplicity of infection (MOI) of 40 in 200 µL of DMEM/F12 containing 10% FBS, followed by incubation for 48 h at 37 °C. Autophagy was observed under a Leica TCS SP8X microscope (Leica, Germany) and evaluated by calculating the number of yellow and red puncta. The number of expressed cells was analyzed using a quantitative digital image analysis system (Image Pro-Plus, version 6.0).
Ad mcherry gfp lc3b adenovirus
Manufactured by Beyotime
Sourced in China
The Ad-mCherry-GFP-LC3B adenovirus is a laboratory tool designed for the study of autophagy. It contains a fusion construct of mCherry, GFP, and the autophagy marker LC3B, which can be used to visualize and monitor autophagic processes in cells.
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11 protocols using ad mcherry gfp lc3b adenovirus
1
Adenoviral Transfection of HPCs for Autophagy Analysis
2
Adenoviral Autophagy Flux Assay
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NPCs were grown on 6-well plates and reached 50–70% confluence at the time of infection. After three washes, cells were infected with Ad-mCherry-GFP-LC3B adenovirus (Beyotime Institute of Biotechnology, Shanghai, China) at a multiplicity of infection of 100 for 24 h. Following treatment, autophagy flux was assessed under a Nikon ECLIPSE Ti microscope (Nikon, Tokyo, Japan).
3
Autophagy Monitoring via mCherry-GFP-LC3B
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Cells were grown on 6-well plates until they reached 20%–30% confluence and then infected with Ad-mCherry-GFP-LC3B adenovirus (Beyotime, Shanghai, China) at an MOI of 10 for 24 h at 37 °C. Based on the manufacture's instruction, the fusion protein of red fluorescent protein mCherry and LC3 can be effectively expressed in target cells after infection, which can be used for autophagy test. Under non-autophagy condition, mCherry-GFP-LC3B exists in the cytoplasm in the form of diffuse yellow fluorescence (the combined effect of mCherry and GFP). Once onset of autophagy, mCherry-GFP-LC3B aggregates on the autophagosome membrane and presents yellow dots. When the autophagosomes fuse with the lysosomes, mCherry-GFP-LC3B presents red spots due to partial quenching of GFP fluorescence in the acidic environment within the lysosomes. At 24 h after niclosamide plus quinacrine treatment, the images were taken with a fluorescence microscope and merged for determining autophagic status of the cells.
4
Monitoring Autophagic Flux with mCherry-GFP-LC3B Adenovirus
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The cells were transfected with Ad-mCherry-GFP-LC3B under non-autophagic conditions, following which the mCherry-GFP-LC3B was present in the cytoplasm, as indicated by dispersive yellow fluorescence. Under conditions of autophagy, mCherry-GFP-LC3B is aggregated on the autophagic membrane, visualized as yellow spots. When the autophagosome is fused with lysosomes, the fluorescence of GFP is quenched, with the reagent presents as red spots. The cells were washed twice with PBS, followed by the addition of 1.5 ml fresh medium and transfected with Ad-mCherry-GFP-LC3B adenovirus (Beyotime Institute of Biotechnology), which adopts a mature E1 defective recombinant adenovirus vector system and expresses the fusion protein of red fluorescent protein mCherry, GFP and LC3B in target cells after infection at a MOI of 20 at 37°C for 24 h. Following Sal B and H2O2 treatment, the variations in LC3B fluorescence were recorded with a fluorescence microscope.
5
Monitoring Autophagy Flux in HepG2 Cells
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HepG2 cells were grown to 40–50% confluence in 12-well plates and transfected with Ad-mCherry-GFP-LC3B adenovirus (#C3011; Beyotime) at an MOI of 50 for 24 h. After two washes, infected HepG2 cells were treated with PA, KYP-2047, or a combination of PA and KYP-2047 for 24 h. After that, autophagy flux was observed under a fluorescence microscope (BX51; Olympus, Tokyo, Japan) and evaluated by calculating the number of yellow and red puncta.
6
Adenoviral Autophagy Imaging Assay
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Cells were grown on 96-well plates until they reached 60%–70% confluence and then transfected with Ad-mCherry-GFP-LC3B adenovirus (Beyotime) at an MOI of 30 for 6 h at 37°C. Following treatment with PP for 18 h, the cells were stained with Hoechst 33342 (Yeasen). Finally, images were taken with an imageXpress® confocal microscope.
7
Autophagy Quantification in AML12 Cells
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AML12 WT and KO Cells were plated on glass slides in 24-well plates. After an overnight attachment, the culture was incubated for 24 h with Ad-mCherry-GFP-LC3B adenovirus (C3011, Beyotime Biotechnology, Shanghai, China). Following the indicated treatments, cells were observed using a confocal microscope (Ultraview VOX, Perkin Elmer, Waltham, MA, USA). The yellow puncta (GFP+mCherry+) represented autophagosomes, whereas the red puncta (GFP−mCherry+) represented autolysosomes. More than 20 images were randomly selected from three independent experiments and analyzed by ImageJ for quantitative comparisons.
8
Quantifying Autophagy with mCherry-GFP-LC3B
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Cells were cultured to 50–60% confluence and transfected with Ad‐mCherry–GFP–LC3B adenovirus (Beyotime, C3011) at an MOI of 30 at 37 °C for 24 h. It is a recombinant adenovirus, which can effectively express fusion proteins of red fluorescent protein mCherry, GFP, and LC3B in target cells after infection. In the case of nonautophagy, mCherry–GFP–LC3B exists in the cytoplasm in the form of diffuse yellow fluorescence (the combined effect of mCherry and GFP). In the case of autophagy, mCherry–GFP–LC3B aggregated on the autophagosome membrane, showing yellow spots. When the autophagosome fuses with the lysosome, the portion of the GFP fluorescence is quenched and appears as a red spot. Following AZD9291 and NPs treatment, LC3B puncta were recorded with a fluorescence microscope and quantified with the ImageJ program (NIH, Bethesda, MD, USA).
9
Monitoring Autophagy via Ad-mCherry-GFP-LC3B
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The Ad-mCherry-GFP-LC3B adenovirus was purchase from Beyotime (C3011, Beyotime Biotechnology, China). HSkMCs were seeded into 12-well plates. When the cells reached 80% confluence, adenovirus Ad-mCherry-GFP-LC3B was added to the cells for 24 h. Then, culture medium containing the virus was removed and replaced with normal medium for 24 h. The immunofluorescence study refers to abovementioned methods.
10
Analyzing Autophagy Flux in L02 Cells
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L02 cells were grown on 24-well plates and reached 50–70% confluence at the time of infection. After two washes, cells were infected with Ad-mCherry-GFP-LC3B adenovirus (Beyotime Institute of Biotechnology, Shanghai, China) at a multiplicity of infection of 100 for 24 h. The infected cells treated with either 400 μM GCDCA or 400 μM TCDCA for 16–32 h. Following indicated treatment, autophagy flux was observed under laser scanning confocal microscope (Leica, Wetzlar, Germany). Autophagy flux was evaluated by calculating the number of yellow and red puncta.
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