LentiBrite GFP-LC3 Lentiviral Biosensor

LentiBrite GFP-LC3 Lentiviral Biosensor

Stock Code: 3586347
Manufacturer Part No: 17-10193
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Application


Research Sub CategoryApoptosis - AdditionalNeurodegenerative Diseases


Fluorescence Microscopy Imaging:
(See Figure 1 in datasheet)
HT-1080 cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 20 for 24 hours. After media replacement and 48 hours further incubation, cells were either left in complete media or incubated for 4 hours in EBSS containing a lysosome inhibitor, to induce autophagy and inhibit lysosomal degradation of autophagosomes.
Cells were fixed with formaldehyde and mounted. Images were obtained by oil immersion wide-field fluorescence microscopy. The GFP-LC3 displays a diffuse nuclear and cytosolic distribution in fed cells, and a punctate distribution in starved autophagic cells.

Immunocytochemistry Comparison and Inhibitor Analysis:
(See Figure 2 in datasheet)
Similar to Figure 1 (see datasheet), HeLa cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 20 for 24 hours. After media replacement and 48 hours further incubation, cells were either left in complete media, incubated for 4 hours in EBSS containing a lysosome inhibitor to induce autophagy and inhibit lysosomal degradation, or incubated as in, with the addition of 5 mM 3-methyladenine (3-MA) as an inhibitor of autophagy. 3-MA completely blocks formation of GFP-LC3-positive autophagic punctae. Immunocytochemical staining (red) of the same fields of view with a monoclonal antibody against LC3A reveals similar expression patterns to the GFP-protein (green), although signal is diminished following 3-MA treatment.

Hard-to-transfect Cell Types:
(See Figure 3 in datasheet)
Primary cell types HUVEC or HuMSC were plated in chamber slides and transduced with lentiviral particles at an MOI of 40 for 24 hours. Subsequent treatments for cells left in complete media or cells incubated in EBSS with lysosome inhibitor, were performed as in Figures 1A and 1B (see datasheet).

Time-lapse Imaging:
(See Figure 5 in datasheet and video online)
HT-1080 cells were treated as in Figure 1B. Shortly following initiation of starvation conditions, time-lapse imaging was performed under temperature-controlled oil immersion wide-field fluorescence microscopy, with images taken every 1 minute over the course of 2 hours. Images demonstrate the translocation of GFP-LC3 from diffuse nuclear/cytosolic localization to discrete cytoplasmic puncta.

Fluorescence Microscopy Imaging:
Cardiomyocytes were cultured on laminin-coated glass bottom dishes in standard medium and transduced with lentiviral particles at a multiplicity of infection of 50. Lentiviral particles carrying a construct of TagGFP2-LC3 driven by the elongation factor-1 promoter (Cat. # 17-10193) were applied for 24 h, the medium was changed and the pharmacological treatment was started after another 24 h. Live images were obtained by using an inverted microscope, 40× objective and a digital camera (Dimitrakis, P., et al. (2012) Cell Tissue Res. 350:361–372).

Quality Level100
ManufacturerSIGMA-ALDRICH
Technique(s)immunocytochemistry: suitable, cell based assay: suitable, transfection: suitable, immunofluorescence: suitable

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