LentiBrite GFP-Tubulin Lentiviral

LentiBrite GFP-Tubulin Lentiviral

Stock Code: 3586353
Manufacturer Part No: 17-10206
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Application


Research Sub CategoryCytoskeleton


Research CategoryCell Structure


Fluorescence microscopy imaging:
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 fixed with formaldehyde and mounted. Image was obtained by oil immersion wide-field fluorescence microscopy. The GFP-tubulin displays predominantly cytoplasmic, fibrillar signal. Able to visualize mitotic cells with visible spindle apparatus formation.

Immunocytochemistry Comparison and Modulator Analysis:
(See Figure 2 in datasheet)
Similar to Figure 1, 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 untreated, incubated for 4 hours with 1 µM paclitaxel (PTX, a microtubule stabilizer), or incubated for 4 hours with 25 µM nocodazole (NZL, a microtubule depolymerization agent). PTX-treated cells become rounded and display the formation of tubulin “bundles”, while NZL eliminates fibrillar tubulin structure. Immunocytochemical staining (red) of the same fields of view with a monoclonal antibody against α-tubulin reveals similar expression patterns to the GFP-protein (green).

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. Cells were either left untreated, or treated for 4 hours with the microtubule stabilizer paclitaxel, at 1 µM concentration.

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.


Components


TagGFP2-Tubulin 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)
MOI = Ratio of # of infectious lentiviral particles (IFU) to # of cells being infected.
Typical MOI values for high transduction efficiency and signal intensity are in the range of 20-40. For this target, some cell types may require lower MOIs (e.g., HT-1080, human umbilical vein endothelial cells (HUVEC)), while others may require higher MOIs (e.g., HeLa, human mesenchymal stem cells (HuMSC), U2OS).
NOTE: MOI should be titrated and optimized by the end user for each cell type and lentiviral target to achieve desired transduction efficiency and signal intensity.


General description

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

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