Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium/F12 (DMEM/F12) were purchased from Gibco (Grand Island, NY, USA). AGE-bovine serum albumin (BSA) was purchased from Merck Millipore (Darmstadt, Germany). BSA and the inhibitor of autophagy 3-methyladenine (3-MA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The inducer of autophagy rapamycin (RA) was purchased from Selleck Chemicals (Houston, TX, USA). Cell Counting Kit-8 (CCK-8) was obtained from Zoman Biotech. Co., Ltd. (Beijing, China). The Annexin V-FITC/PI apoptosis detection kit was purchased from Multi Sciences Biotech. Co., Ltd. (Hangzhou, China). The mCherry-GFP-LC3b-adenovirus was purchased from Beyotime Institute of Biotechnology (Shanghai, China). The GAPDH antibody was obtained from Abcam (Cambridge, MA, USA). Light chain (LC) 3B, Beclin1, MMP-3, and MMP-13 antibodies were purchased from Novus Biologicals (Littleton, CO, USA).
Mcherry gfp lc3b adenovirus
Manufactured by Beyotime
Sourced in China
MCherry-GFP-LC3B adenovirus is a recombinant adenoviral vector that expresses a fluorescently labeled autophagy marker protein, LC3B, fused with mCherry and GFP. This construct is designed for the study of autophagy dynamics in living cells.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco s modified eagle s medium f12 dmem f12, by Thermo Fisher Scientific (1 mentions)
Beclin 1, by Novus Biologicals (1 mentions)
Mmp 13, by Novus Biologicals (1 mentions)
Age bovine serum albumin, by Merck Group (1 mentions)
Gapdh antibody, by Abcam (1 mentions)
3 methyladenine 3 ma, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by MultiSciences Biotech (1 mentions)
Laser confocal microscopy, by Zeiss (1 mentions)
Laser scanning confocal microscope, by Nikon (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Polybrene, by Beyotime (1 mentions)
Confocal system, by Nikon (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Fluorescence microscope, by Leica camera (1 mentions)
Dmi3000b, by Leica camera (1 mentions)
7 protocols using mcherry gfp lc3b adenovirus
1
Autophagy Regulation in Cellular Processes
2
Autophagic Flux Monitoring in Hepatocytes
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To detect the autophagic flux, hepatocytes were transfected with mCherry-GFP-LC3B adenovirus (C3011, Beyotime Institute of Biotechnology, Beijing, China). The hepatocytes were grown on 24-well plates until they reached approximately 30–50% confluence at the time of infection. Briefly, the hepatocytes were transfected with the mCherry-GFP-LC3B adenovirus at an MOI of 10 for 36 h at 37 °C before treatment with NEFA. After treatment with NEFA for 5 h, the hepatocytes were fixed with 4% paraformaldehyde and analyzed using laser confocal microscopy (Carl Zeiss GmbH, Jena, Germany).
3
Analyzing Autophagy Flux via mCherry-GFP-LC3B
4
mCherry-GFP-LC3B Assay for Autophagy
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For the mCherry-GFP-LC3B assay, HK-2 cells were seeded in 6-well plates on microscope glass coverslips, infected with mCherry-GFP-LC3B adenovirus (Beyotime) for 24 h, and treated with NG or HG, CQ and ICA for another 48 h. Following treatment, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and incubated with DAPI (1:1,000) for 10 min, then examined by confocal microscopy (CLSM, Carl Zeiss LSM800, Jena, Germany).
5
Autophagy Modulation in Primary GCs
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Primary GCs of 2 × 104 were seeded on coverslips in a 24-well plate and incubated for 12 h at 37 °C and 5% CO2 before adding the mixture of mCherry-GFP-LC3B adenovirus (Beyotime, Shanghai, China) and pro-viral infection reagent Polybrene (Beyotime) for 24 h. After replacing with fresh medium, cells were treated with CTX, VEGFA, 3-MA, or/and rapamycin. The treated cells were fixed with 4% paraformaldehyde for 10 min and stained with DAPI for 10 min. After washing, the cells were sealed with an antifade solution and visualized using the confocal system (Nikon).
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Primary GCs of 2 × 104 were seeded on coverslips in a 24-well plate and incubated for 12 h at 37 °C and 5% CO2 before adding the mixture of mCherry-GFP-LC3B adenovirus (Beyotime, Shanghai, China) and pro-viral infection reagent Polybrene (Beyotime) for 24 h. After replacing with fresh medium, cells were treated with CTX, VEGFA, 3-MA, or/and rapamycin. The treated cells were fixed with 4% paraformaldehyde for 10 min and stained with DAPI for 10 min. After washing, the cells were sealed with an antifade solution and visualized using the confocal system (Nikon).
6
Visualization of Autophagy in RA-FLSs
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RA-FLSs were plated in 12-well plates at a density of 5× 104/well. Twelve hours after plating, the mCherry-GFP-LC3B adenovirus (Beyotime Institute of Biotechnology, Shanghai, China) at 20 multiplicity of infection (MOI) was added to the cells according to the manufacturer’s protocol. Twenty-four hours after infection, the RA-FLSs were transfected with DERL1-siRNA (80 nM) or negative control siRNA (80 nM) using Lipofectamine 2000 according to the manufacturer’s recommendations. Cells were then cultured for another 48 hours, and 0.1 μM of bafilomycin A1 (Sigma-Aldrich, St. Louis, MO, USA) or matched amounts of dimethyl sulfoxide (DMSO) were added to cell cultures for the last 4 hours, and then photographed under a fluorescence microscope (Leica, Germany).
7
Sarmentosin-Induced Autophagy in HepG2 Cells
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HepG2 cells were cultured and seeded on 24-well plates (Corning, #3524) containing sterile glass slides, with 20,000 cells per well. According to the instructions, HepG2 cells were transfected with Nrf2 siRNA and mCherry-GFP-LC3B adenovirus (Beyotime, #C3012). After incubating for 1 h with or without 3-MA (10 mM), CQ (20 µM), BafA1 (10 nM), or rapamycin (10 nM), the cells were coincubated with 1640 single medium containing 20 µM sarmentosin for 6 or 12 h. HepG2 cells were washed with PBS, and the cells were fixed with 4% paraformaldehyde for 30 min at room temperature. Subsequently, the glass slide containing the cells was removed, and anti-fluorescence quenching mounting solution was added dropwise. A fluorescence microscope (Leica, DMI3000B) was used to take pictures.
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