NFκB-TIME

Both NFkB-TIME (CRL-4049) and its parental cell line TIME (CRL-4025) exhibit normal diploid karyotype, extended lifespan in culture and endothelial characteristics that make them ideal models for investigating many aspects of endothelial cell biology.  

NFkB-TIME (ATCC CRL-4049) retains angiogenic potential and the expression of NanoLuc® luciferase under the control of multiple copies of the NFkB response element.  When the NFkB-TIME (ATCC CRL-4049) cells are exposed to inflammatory cytokine such as TNFα, activation of the NFkB signaling pathway occurs resulting in increased NanoLuc® luciferase activity.  The high sensitivity, excellent signal/background ratio and simple single-addition assay makes this reporter cell line an ideal replacement for the cumbersome and highly variable ICAM-1/CD54 activation assays.

TIME (ATCC CRL-4025) cells are transfected with linearized pNL3.2.NF-κB-RE[NlucP/NF-κB-RE/Hygro] plasmid, which expresses the NanoLuc® luciferase under the control of multiple copies of the NFkB response element.   A stable clone with high activation of the NanoLuc® luciferase activity upon exposure to inflammatory cytokine TNFα is selected to establish the NFkB-TIME cell line.

TIME (ATCC CRL-4025) cells are transfected with linearized pNL3.2.NF-κB-RE[NlucP/NF-κB-RE/Hygro] plasmid, which expresses the NanoLuc® luciferase under the control of multiple copies of the NFkB response element.   A stable clone with high activation of the NanoLuc® luciferase activity upon exposure to inflammatory cytokine TNFα is selected to establish the NFkB-TIME cell line.

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

1. Prepare a 25 cm² or a 75 cm² culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents, the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
2. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
3. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All operations from this point on should be carried out under strict aseptic conditions.
4. Transfer the vial contents to a centrifuge tube containing 9.0 mL of complete culture medium and centrifuge the cell suspension at approximately 125 x g for 5 to 7 minutes.
5. Discard the supernatant and resuspend the cells in fresh growth medium (see the batch-specific information for the recommended dilution ratio). Add this suspension to the prepared culture vessel.
6. Incubate the culture at 37°C in a suitable incubator.

1. Remove and discard spent medium.
2. Briefly rinse the cells with Dulbecco's Phosphate Buffered Saline (DPBS, ATCC 30-2200) and discard rinse solution.
3. Add 2.0 to 3.0 mL room temperature Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) to the flask. Incubate at 37°C for 5 min (until cells have detached).
4. Neutralize trypsin by adding an equal volume of room temperature 2% FBS in DPBS.
5. Centrifuge cells at 250 x g for 5 min at room temperature.
6. Remove supernatant. Resuspend pellet in 6.0 to 8.0 mL Complete Growth Medium.
7. Count cells, and seed 5.0 x 10³ to 8.0 x 10³ viable cells/cm² to new culture vessels.

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