LentiBrite RFP-p62 Lentiviral Biosensor

LentiBrite RFP-p62 Lentiviral Biosensor

Stock Code: 3586386
Manufacturer Part No: 17-10404
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Application


Fluorescence Microscopy
Imaging:
(See Figure 1 in datasheet)
Primary cell type, Human mesenchymal stem cells (HuMSC), were plated in a chamber slide and transduced with lentiviral particles at an MOI of 40 for 24 hours. After media replacement and 24 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 RFP-p62 displays a diffuse cytosolic distribution with scattered small puncta in fed cells, and an entirely punctate distribution in starved cells.


Immunocytochemistry Comparison:
(See Figure 2 in datasheet)
Similar to Figure 1 (see datasheet), U2OS cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 40 for 24 hours. After 24 hours, media was replaced and cells were then further incubated for 24 hours. Cells were then fixed and stained with a polyclonal antibody against p62 (Cat. No. MABC32) followed by a FITC-labeled anti-rabbit IgG. Distribution of the RFP-p62 (red) is similar to that detected by immunocytochemical staining (green).

Hard-to-transfect Cell Types:
(See Figure 3 in datasheet)
Primary cell type HUVEC were plated in a chamber slide 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)
U2OS cells were plated in coverglass chamber slides and transduced with RFP-p62 lentiviral particles as in Figure 1 (see datasheet). After replacing growth media with EBSS containing a lysosomal inhibitor, images were collected every 20 seconds for a total of 32 min. The RFP-p62 displays a diffuse cytosolic distribution in fed cells, and a punctate distribution in starved autophagic cells.

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


TagRFP-p62 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.
Promoter
EF-1 (Elongation Factor-1)
Multiplicty of Infection (MOI)

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

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