The tandem mRFP-GFP-LC3 adenoviruses construct was obtained from Hanbio Inc (Shanghai, China) and was used to evaluate autophagy induction. Briefly, 5 × 104 PLC/PRF/5 or HLE cells cultured on coverslips in 24- well microplates were transduced with mRFP-GFP-LC3 adenoviruses at 50 MOI. Two hours after transduction, removed the adenovirus by washing the cells with PBS, and then maintained the cells in complete culture medium with 40 μM ATRA or vehicle for 24 h. The cells were washed with PBS, fixed with 4% paraformaldehyde, and viewed under a fluorescence microscope. The number of mRFP, GFP and GFP-mRFP (yellow) dots were determined by manual counting of fluorescent puncta in five fields from three different cells. The number of dots per cell was obtained by dividing the total number of dots by the number of cell in each microscopic field.
Tandem mrfp gfp lc3 adenoviruses construct
Manufactured by Hanbio Biotechnology
The Tandem mRFP-GFP-LC3 adenoviruses construct is a tool used for the visualization and quantification of autophagy. It contains a fusion of mRFP (monomeric red fluorescent protein) and GFP (green fluorescent protein) tags linked to the autophagy marker protein LC3 (microtubule-associated protein 1A/1B-light chain 3). This construct allows for the real-time monitoring of autophagic flux in living cells.
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2 protocols using tandem mrfp gfp lc3 adenoviruses construct
1
Quantifying Autophagy Induction with mRFP-GFP-LC3
2
Visualizing Autophagy Induction via mRFP-GFP-LC3
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The tandem mRFP-GFP-LC3 adenoviruses construct was obtained from Hanbio Inc (Shanghai, China)
and was used to evaluate autophagy induction. Brie y, 5 × 10 4 PLC/PRF/5 or HLE cells cultured on coverslips in 24-well microplates were transduced with mRFP-GFP-LC3 adenoviruses at 50 MOI. Two hours after transduction, removed the adenovirus by washing the cells with PBS, and then maintained the cells in complete culture medium with 40µM ATRA or vehicle for 24 h. The cells were washed with PBS, xed with 4% paraformaldehyde, and viewed under a uorescence microscope. The number of mRFP, GFP and GFP-mRFP (yellow) dots were determined by manual counting of uorescent puncta in ve elds from three different cells. The number of dots per cell was obtained by dividing the total number of dots by the number of cell in each microscopic eld.
and was used to evaluate autophagy induction. Brie y, 5 × 10 4 PLC/PRF/5 or HLE cells cultured on coverslips in 24-well microplates were transduced with mRFP-GFP-LC3 adenoviruses at 50 MOI. Two hours after transduction, removed the adenovirus by washing the cells with PBS, and then maintained the cells in complete culture medium with 40µM ATRA or vehicle for 24 h. The cells were washed with PBS, xed with 4% paraformaldehyde, and viewed under a uorescence microscope. The number of mRFP, GFP and GFP-mRFP (yellow) dots were determined by manual counting of uorescent puncta in ve elds from three different cells. The number of dots per cell was obtained by dividing the total number of dots by the number of cell in each microscopic eld.
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