LentiBrite GFP-Vimentin Lentiviral

LentiBrite GFP-Vimentin Lentiviral

Stock Code: 3586339
Manufacturer Part No: 17-10152
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Application


Immunocytochemistry Comparison Analysis:
Similar to Figure 1 in the datasheet, HeLa cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 20 for 24 hours. After 24 hours, media was replaced and cells were then further incubated for 48 hours. Immunocytochemical staining (red) of the same fields of view with a monoclonal antibody against Vimentin (Cat No. MAB3400) reveals similar expression patterns to the GFP-protein (green).

Hard-to-transfect Cell Types:
Primary cell types HUVEC or HuMSC were plated in chamber slides and transduced with lentiviral particles at an MOI of 10 for 24 hours.

Confocal Microscopy Imaging:
HeLa and U2OS cells were treated as in Figure 2A in the datasheet. Images were obtained by oil immersion confocal fluorescence microscopy.

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-Vimentin 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 “Viral Titer” in 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, U2OS), while others may require higher MOIs (e.g., human mesenchymal stem cells (HuMSC), human umbilical vein endothelial cells (HUVEC ), HeLa).
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


Intermediate filaments (IFs) are 10 nm diameter cytoskeletal structures that typically form a cage-like structure extending from the nucleus to the plasma membrane. The five families of IF proteins have distinct polymer characteristics and cell type-specific expression patterns. Expression of vimentin, a type-III IF protein, is restricted to mesenchymal cells, and appearance of vimentin in epithelial cancer cells is a hallmark of the epithelial-to-mesenchymal transition. Vimentin assembles into classic long filaments and short filaments termed squiggles. Time-lapse microscopy of live cells expressing GFP-vimentin has revealed the dynamics of vimentin IF maturation, and crosstalk with microtubules and microfilaments.

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

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