KC-4317

TF-1-NFκB-Luc2-DR3-Cell-Line

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Background of TF-1-NFκB-Luc2-DR3-Cell-Line

TL1A and its functional receptor DR3 are members of the TNF/TNFR superfamilies of proteins.It is expressed preferentially in the tissues enriched in lymphocytes, and it may play a role in regulating lymphocyte homeostasis. This receptor has been shown to stimulate NF-kappa B activity and regulate cell apoptosis. The signal transduction of this receptor is mediated by various death domain containing adaptor proteins.NF-κB pathway consists of canonical and non-canonical pathways. The canonical NF-κB is activated by various stimuli, transducing a quick but transient transcriptional activity, to regulate the expression of various proinflammatory genes and also serve as the critical mediator for inflammatory response. Meanwhile, the activation of the non-canonical NF-κB pathway occurs through a handful of TNF receptor superfamily members.

Specifications

Catalog NumberKC-4317
Cell Line NameTF-1-NFκB-Luc2-DR3-Cell-Line
Host Cell LineTF-1-NFκB-Luc2
DescriptionStable TF-1 cell line expressing exogenous luciferase gene under the control of DR3 signaling pathway
QuantityTwo vials of frozen cells (≥2-106/vial)
StabilityStable in culture over a minimum of 10 passages
ApplicationDrug screening and biological assays
Freezing MediumRPMI1640 + 20% FBS + 10% DMSO
Propagation MediumRPMI1640 + 10% FBS+5ng/mL rhGM-CSF+100μg/mL Hygromycin B+1μg/mL puromycin
Selection MarkerHygromycin B and puromycin
MorphologyMostly single, round (some polymorph) cells in suspension
SubcultureSplit saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 105 cells/mL
Incubation37 °C with 5% CO2
StorageLiquid nitrogen immediately upon receiving
Doubling TimeApproximately 30 hours
Mycoplasma StatusNegative
In Vivo ValidationNA

Cell Line Generation

TF-1-NFκB-Luc2-DR3 cell line was generated using lentivirus expressing DR3 sequence.

Characterization

Figure 1. TF-1-NFκB-Luc2-DR3 cell line was seeded into the 96-well plate, and treated with TL1A at a maximum concentration of 1μg/mL for 6 hours, then readout with Bright-Glo system.

Figure 2. TF-1-NFκB-Luc2-DR3 cell line was seeded into the 96-well plate, and treated with PF-06480605-hIgG1 at a maximum concentration 20μg/mL for 1 hours, then treated with TL1A (100ng/mL), incubated for 6 hours and readout with Bright-Glo system.

Cell Resuscitation

  1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS, 5ng/mL rhGM-CSF, 100μg/mL Hygromycin and 1μg/mL Puromycin)in a 37°C water bath.
  2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
  3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
  4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
  5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
  6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
  7. Incubate the flask at 37°C, 5% CO2 incubator.
  8. Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 105 cells/mL.

Cell Freezing

  1. Prepare the freezing medium (70% RPMI1640 + 20% FBS + 10% DMSO) fresh immediately before use.
  2. Keep the freezing medium on ice and label cryovials.
  3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
  4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
  5. Resuspend the cells at a density of at least 3×106 cells/mL in chilled freezing medium.
  6. Aliquot 1 mL of the cell suspension into each cryovial.
  7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
  8. Transfer vials to liquid nitrogen for long-term storage.

References

  1. Valatas V, Kolios G, Bamias G. TL1A (TNFSF15) and DR3 (TNFRSF25): A Co-stimulatory System of Cytokines With Diverse Functions in Gut Mucosal Immunity. Front Immunol. 2019 Mar 27;10:583. doi: 10.3389/fimmu.2019.00583. PMID: 30972074; PMCID: PMC6445966.
  2. Chinnaiyan AM, O'Rourke K, Yu GL, Lyons RH, Garg M, Duan DR, Xing L, Gentz R, Ni J, Dixit VM. Signal transduction by DR3, a death domain-containing receptor related to TNFR-1 and CD95. Science. 1996 Nov 8;274(5289):990-2. doi: 10.1126/science.274.5289.990. PMID: 8875942.
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