LentiBrite Calreticulin-RFP-KDEL L

LentiBrite Calreticulin-RFP-KDEL L

Stock Code: 3586337
Manufacturer Part No: 17-10146
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Application


Research CategoryApoptosis & Cancer


Research Sub CategoryApoptosis - Additional


Fluorescence Microscopy Imaging:
(See Figure 1 in datasheet)
U2OS cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 5 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 calreticulin-RFP-KDEL appears in a meshwork pattern in the cytoplasm.

Immunocytochemistry Comparison and Inhibitor Analysis:
(See Figure 2 in datasheet)
Similar to Figure 1, HeLa cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 40 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 polyclonal antibody against calreticulin reveals similar expression patterns to the RFP-protein (red).

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

Additional Cell Type:
(See Figure 4 in datasheet)
HT1080 and REF52 cells were treated as in Figure 1.

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


Calreticulin-TagRFP-KDEL 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-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), while others may require higher MOIs (e.g., human mesenchymal stem cells (HuMSC), 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, immunofluorescence: suitable

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