LentiBrite GFP-LC3 Control Mutant

LentiBrite GFP-LC3 Control Mutant

Stock Code: 3586345
Manufacturer Part No: 17-10189
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Application


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 lleft 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 Control Mutant displays a diffuse nuclear and cytosolic distribution in both fed and starved autophagic cells (i.e., no translocation to a punctate cytoplasmic distribution as characteristic of wild-type LC3).

Immunocytochemistry Comparison and Inhibitor Analysis:
(See Figure 2 in datasheet)
Similar to Figure 1 conditions (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 does not affect GFP-LC3 Control Mutant localization. Immunocytochemical staining (red) of the same fields of view with a monoclonal antibody against LC3A reveals a similar expression pattern to the mutant protein (green) under fed conditions. Immunostaining of starved cells displays the punctate distribution of endogenous wild-type LC3, while signal following 3-MA treatment is diminished.

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).

For optimal fluorescent visualization, it is recommended to analyze the target expression level within 24-48 hrs after transfection/infection for optimal live cell analysis, as fluorescent intensity may dim over time, especially in difficult-to-transfect cell lines. Infected cells may be frozen down after successful transfection/infection and thawed in culture to retain positive fluorescent expression beyond 24-48 hrs. Length and intensity of fluorescent expression varies between cell lines. Higher MOIs may be required for difficult-to-transfect cell lines.


Research Sub CategoryApoptosis - AdditionalNeurodegenerative Diseases


Research CategoryApoptosis & CancerNeuroscience


Components


TagGFP2-LC3-G120A (Mutant) Lentivirus: One vial containing 25 µL of lentiviral particles at a minimum of 3 x 10E8 infectious units (IFU) per mL. For lot-specific titer information, please see lot specific “Viral Titer” in the product specifications of the datasheet.PromoterEF-1 (Elongation Factor-1)Multiplicty of Infection (MOI)MOI = Ratio of # of infectious lentiviral particles (IFU) to # of cells being infected

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

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