LentiBrite alpha-actinin-GFP Lentivira

LentiBrite alpha-actinin-GFP Lentivira

Stock Code: 3586343
Manufacturer Part No: 17-10156
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Application


Fluorescence Microscopy Imaging:
(See Figure 1 in datasheet)
REF52 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. Images were obtained by oil immersion wide-field fluorescence microscopy. The α-actinin-GFP appears in a periodic distribution along microfilamentous structures.

Additional Cell Types:
(See Figure 2 in datasheet)
U2OS and HT1080 cells were treated as in Figure 1 (see datasheet) at an MOI of 40 and 10, respectively, for 24 hours.

Hard-to-transfect Cell Types:
(See Figure 3 in datasheet)
Primary cell types HUVEC and Human mesenchymal stem cells (HuMSC) were plated in chamber slides and transduced with lentiviral particles at an MOI of 40 and 10, respectivley, for 24 hours.

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 CategoryCell Structure


Research Sub CategoryAdhesion (CAMs)


Components


α-actinin-TagGFP 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 box above.Promoter
EF-1 (Elongation Factor-1Multiplicty 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, HeLa, human mesenchymal stem cells (HuMSC)), while others may require higher MOIs (e.g., human umbilical vein endothelial cells (HUVEC), 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


Migration of cells requires engagement of sites of extracellular matrix contact (focal adhesions) with the actin cytoskeleton. Contractile force is further generated when the actin microfilaments develop into stress fibers by bundling in an anti-parallel fashion and recruiting myosin II. A key component of stress fibers is the spectrin-related protein, α-actinin, which directly links actin microfilaments and also couples them to focal adhesions. Imaging of live cells containing α-actinin fused with fluorescent proteins has provided key insights into the dynamic mechanisms of stress fiber assembly and adaptation to strain.

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

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