LentiBriteTagRFP-Rad51 LentiviralBiosnsr

LentiBriteTagRFP-Rad51 LentiviralBiosnsr

Stock Code: 3586357
Manufacturer Part No: 17-10220
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Application


Research CategoryApoptosis & Cancer


Fluorescence Microscopy Imaging:
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 fixed with formaldehyde and mounted. Images were obtained by oil immersion wide-field fluorescence microscopy. The RFP-Rad51 displays a variably punctate distribution in the nucleus.

Immunocytochemistry Comparison:
U2OS 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) with a polyclonal antibody against Rad51 (Cat No. ABE257) reveals a similar expression pattern to RFP-Rad51 (red).

Hard-to-transfect Cell Types:
Primary cell types HUVEC or HuMSC were plated in chamber slides and transduced with RFP-Rad51 lentiviral particles at an MOI of 20 and 5, respectively, for 24 hours.

Confocal Microscopy Imaging:
U2OS cells were transduced with RFP-Rad51 lentivirus. Cells were counterstained with FITC-phalloidin to show the actin filaments (green) and DAPI to show the nucleus (blue). Images were obtained by oil immersion confocal fluorescence microscopy.

Additional Cell Type:
HT1080 cells were treated.

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 Sub CategoryCell Cycle, DNA Replication & Repair


Components


TagRFP-Rad51 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, HeLa, U2OS, human mesenchymal stem cells (HuMSC)), while others may require higher MOIs (e.g., human umbilical vein endothelial cells (HUVEC)).
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


Read our application note in Nature Methods!http://www.nature.com/app_notes/nmeth/2012/121007/pdf/an8620.pdf(Click Here!)Learn more about the advantages of our LentiBrite Lentiviral Biosensors! Click Here

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

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