LentiBrite Paxillin-RFP Lentiviral

LentiBrite Paxillin-RFP Lentiviral

Stock Code: 3586342
Manufacturer Part No: 17-10155
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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 40 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 paxillin-RFP displays a punctuate distribution at focal adhesions in cells.

Immunocytochemistry comparison and inhibitor analysis:
(See Figure 2 in datasheet)
Similar to Figure 1 (see datasheet), HeLa cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 5 for 24 hours. After 24 hours, media was replaced and cells were then further incubated for 48 hours. Immunocytochemical staining (green) of the same fields of view with a monoclonal antibody against paxillin (Cat No. 05-417) reveals similar expression patterns to the protein-RFP (red).

Additional cell types:
(See Figure 3 in datasheet)
U2OS and HT1080 cells were treated as in Figure 1 (see datasheet) at an MOI of 5 and 10, respectively, for 24 hours.

Hard-to-transfect cell types:
(See Figure 4 in datasheet)
Primary cell type HUVEC and Human mesenchymal stem cells (HuMSC) were plated in chamber slides and transduced with lentiviral particles at an MOI of 5 and 20, repectively, 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


Paxillin-TagRFP 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. 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 mesenchymal stem cells (HuMSC)), while others may require higher MOIs (e.g., human umbilical vein endothelial cells (HUVEC), U2OS, 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


Adherent cells maintain contact with the extracellular substratum by means of focal adhesions, comprising transmembrane matrix receptors (integrins) linked on their cytoplasmic side to a complex of signaling and structural proteins, which are in turn linked to the cytoskeleton

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

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