LentiBrite GFP-beta-actin Lentiviral

Stock Code: 3586352
Manufacturer Part No: 17-10204

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Descriptions

Application

Research Sub CategoryCytoskeleton

Research CategoryCell Structure

Immunocytochemistry comparison and inhibitor 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 media replacement and 48 hours further incubation, cells were either untreated, incubated for 2 hours in complete media containing 2µM cytochalasin-D, or incubated for 2 hours in complete media containing 200nM jasplakinolide to induce actin depolymerization. Immunocytochemical staining (red) of the same fields of view with TRITC-conjugated Phalloidin 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 40 for 24 hours.

Confocal microscopy imaging: HeLa cells were treated as in Figure 2A in the datasheet. Cells were then treated using ProteoExtract™ Native Cytoskeleton Enrichment Kit (Cat No. 17-10210). Unenriched & Enriched. Images were obtained by oil immersion confocal fluorescence microscopy.

HT-1080 cells were treated as in Figures 2A and 2B in the datasheet.
Confocal microscopy imaging: HT1080 cells were treated as in Figures 2A and 2B.

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-β-actin 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 product specifications above. PromoterEF-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., human mesenchymal stem cells (HuMSC), HeLa, 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

See a time-lapse video of live cells undergoing actin depolymerization using the LentiBrite GFP-Beta Actin Lentiviral Biosensor!Click HereRead our application note in Nature Methods!http://www.nature.com/app_notes/nmeth/2012/121007/pdf/an8620.pdf(Click Here!)

Specifications

Quality Level	100
Manufacturer	SIGMA-ALDRICH
Technique(s)	immunocytochemistry: suitable, cell based assay: suitable, transfection: suitable, single cell analysis: suitable, immunofluorescence: suitable