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Current position:Product Center > Cell lines > WNT > wnt > TCF LEF Reporter HEK-293 Cell Line
TCF LEF Reporter HEK-293 Cell Line
Description
Cat. No: GM-C29875
Product: TCF LEF Reporter HEK-293 Cell Line


β-catenin is an important cellular protein involved in cell adhesion and signal transduction. At the cell membrane, β-catenin binds to cadherins to form adherens junctions and connects to the cytoskeleton. At the same time, β-catenin is also a key component of the WNT signaling pathway. Its stability and activity are regulated by the destruction complex and various signaling pathways. In the canonical WNT signaling pathway, when WNT proteins (such as Wnt3a) activate Frizzled receptors and their co-receptor LRP5/6, the destruction complex is inhibited, allowing β-catenin to stabilize and accumulate in the cytoplasm. It then translocates into the nucleus, where it binds to TCF/LEF transcription factors to activate the expression of genes related to cell proliferation, differentiation, and survival.

TCF LEF Reporter HEK-293 Cell Line is a clonal stable HEK-293 cell line constructed using lentiviral technology, with signal-dependent expression of a luciferase reporter gene. The Wnt3a protein activates Frizzled receptors and their co-receptors, inhibiting the degradation of β-catenin, promoting its translocation into the nucleus, where it binds to TCF/LEF transcription factors, activates downstream signaling pathways, and induces luciferase expression. By measuring luciferase activity, the activation level of the signaling pathway can be sensitively indicated, making it useful for evaluating the in vitro effects of WNT-related drugs.
Data
Response to Recombinant Human Wnt-3a Protein. The TCF LEF Reporter HEK-293 Cell Line (Cat. GM-C29875) at a concentration of 1.5E4 cells/well (96-well format) was stimulated with serial dilutions of Recombinant Human Wnt-3a Protein (R&D SYSTEMS/5036-WN/CF) in assay buffer (DMEM + 1% FBS + 1% P.S) for 16 hours. The firefly luciferase activity was measured using the GMOne-Step Luciferase Reporter Gene Assay Kit (Cat. GM-040503). The maximum induction fold was approximately [113.3]. Data are shown by drug mass concentration.
Response to Human DKK-1 Protein. Serial dilutions of the Human DKK-1 Protein (SinoBiological/10170-H08H) was incubated with 1.5E4 cells/well of the TCF LEF Reporter HEK-293 Cell Line (Cat. GM-C29875) in a 96-well plate for 1 hour in assay buffer (DMEM+1% FBS+1% P.S). Subsequently, the recombinant human Wnt-3a (R&D/5036-WN-CF) at a concentration of 15 ng/well was added, and the coculture proceeded for an additional 16 hours. Firefly luciferase activity was then measured using the GMOne-Step Luciferase Reporter Gene Assay Kit (Cat. GM-040503). The results indicated maximum blocking folds of approximately [52.3]. Data are shown by drug mass concentration.
The passage stability of response to Recombinant Human Wnt-3a Protein. The passage 5 and 27 of TCF LEF Reporter HEK-293 Cell Line (Cat. GM-C29875) at a concentration of 1.5E4 cells/well (96-well format) were stimulated with serial dilutions of Recombinant Human Wnt-3a Protein (R&D SYSTEMS/5036-WN/CF) in assay buffer (DMEM + 1% FBS + 1% P.S) for 16 hours. The firefly luciferase activity was measured using the GMOne-Step Luciferase Reporter Gene Assay Kit (Cat. GM-040503). Data are shown by drug mass concentration.
Specifications
Cat. NoGM-C29875
ProductTCF LEF Reporter HEK-293 Cell Line
Product Format1 vial of frozen cells
Quantity5E6 Cells per vial,1 mL
Storage ConditionsLiquid nitrogen immediately upon receipt
Recovery MediumDMEM+10% FBS+1% P.S
Growth mediumDMEM+10% FBS+1% P.S+4 μg/mL Blasticidin
NoteNone
Freezing Medium90% FBS+10% DMSO
Growth propertiesAdherent
Growth Conditions37°C, 5% CO₂
Safety considerationsBiosafety Level 2
Mycoplasma TestingThe cell line has been screened to confirm the absence of Mycoplasma species.
Materials
ReagentOrdering Information
DMEMGibco/C11995500BT
Fetal Bovine SerumCegrogen biotech/A0500-3010
Pen/StrepThermo/15140-122
BlasticidinGenomeditech/GM-040404
Recombinant Human Wnt-3a ProteinR&D SYSTEMS/5036-WN
Recombinant Human DKK-1 Protein (His Tag), HPLC-verifiedSino Biological/10170-H08H
GMOne-Step Luciferase Reporter Gene Assay KitGenomeditech/GM-040503
Cell Culture

Cell Recovery

Recovery Medium: DMEM+10% FBS+1% P.S

To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C. Storage at -70°C will result in loss of viability.

a)       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 - 3 minutes).

b)       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 of the operations from this point on should be carried out under strict aseptic conditions.

c)       Transfer the vial contents to a centrifuge tube containing 5.0 mL complete culture medium and spin at approximately 176 x g for 5 minutes. Discard supernatant.

d)       Resuspend cell pellet with the recommended recovery medium. And dispense into appropriate culture dishes.

e)       Incubate the culture at 37°C in a suitable incubator. A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.

Cell Freezing

Freezing Medium: 90% FBS+10% DMSO

a)          Centrifuge at 176 x g for 3 minutes to collect cells.

b)         Resuspend the cells in pre-cooled freezing medium and adjust the cell density to 5E6 cells/mL.

c)          Aliquot 1 mL into each vial.

d)         Place the vial in a controlled-rate freezing container and store at -80°C for at least 1 day, then transfer to liquid nitrogen as soon as possible.

Cell passage

Growth medium: DMEM+10% FBS+1% P.S+4 μg/mL Blasticidin

For the first 1 to 2 passages post-resuscitation, use the recovery medium. Once the cells have stabilized, switch to a growth medium.

a)          Subculturing is necessary when the cell density reaches 80%. It is recommended to perform subculturing at a ratio of 1:3 to 1:4 every 2-3 days. Ensure that the density does not exceed 80%, as overcrowding can lead to reduced viability due to compression.

b)         Remove and discard culture medium.

c)          Briefly rinse the cell layer with PBS to remove all traces of serum that contains trypsin inhibitor.

d)         Add 1.0 mL of 0.25% (w/v) Trypsin-EDTA solution to dish and observe cells under an inverted microscope until cell layer is dispersed (usually within 30 to 60 seconds at 37°C).

e)          Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.

f)          Add 2.0 mL of growth medium to mix well and aspirate cells by gently pipetting.

g)         After centrifugation, resuspend the pellet and add appropriate aliquots of the cell suspension to new culture vessels.

h)         Incubate cultures at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:3 - 1:4 is recommended

Medium Renewal: Every 2 to 3 days

Notes

a)          Upon initial thawing, a higher number of dead cells is observed, which is a normal phenomenon. Significant improvement is seen after adaptation. Once the cells reach a stable state, the number of dead cells decreases after subculturing and the cell growth rate becomes stable.

b)         Ensure that the cell density does not exceed 80%, as overcrowding may lead to reduced viability due to compression.

 


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Current Position:Product Center > Cell lines > WNT > wnt > TCF LEF Reporter HEK-293 Cell Line
classify
TCF LEF Reporter HEK-293 Cell Line
Description
Cat. No: GM-C29875
Product: TCF LEF Reporter HEK-293 Cell Line


β-catenin is an important cellular protein involved in cell adhesion and signal transduction. At the cell membrane, β-catenin binds to cadherins to form adherens junctions and connects to the cytoskeleton. At the same time, β-catenin is also a key component of the WNT signaling pathway. Its stability and activity are regulated by the destruction complex and various signaling pathways. In the canonical WNT signaling pathway, when WNT proteins (such as Wnt3a) activate Frizzled receptors and their co-receptor LRP5/6, the destruction complex is inhibited, allowing β-catenin to stabilize and accumulate in the cytoplasm. It then translocates into the nucleus, where it binds to TCF/LEF transcription factors to activate the expression of genes related to cell proliferation, differentiation, and survival.

TCF LEF Reporter HEK-293 Cell Line is a clonal stable HEK-293 cell line constructed using lentiviral technology, with signal-dependent expression of a luciferase reporter gene. The Wnt3a protein activates Frizzled receptors and their co-receptors, inhibiting the degradation of β-catenin, promoting its translocation into the nucleus, where it binds to TCF/LEF transcription factors, activates downstream signaling pathways, and induces luciferase expression. By measuring luciferase activity, the activation level of the signaling pathway can be sensitively indicated, making it useful for evaluating the in vitro effects of WNT-related drugs.
Data
Response to Recombinant Human Wnt-3a Protein. The TCF LEF Reporter HEK-293 Cell Line (Cat. GM-C29875) at a concentration of 1.5E4 cells/well (96-well format) was stimulated with serial dilutions of Recombinant Human Wnt-3a Protein (R&D SYSTEMS/5036-WN/CF) in assay buffer (DMEM + 1% FBS + 1% P.S) for 16 hours. The firefly luciferase activity was measured using the GMOne-Step Luciferase Reporter Gene Assay Kit (Cat. GM-040503). The maximum induction fold was approximately [113.3]. Data are shown by drug mass concentration.
Response to Human DKK-1 Protein. Serial dilutions of the Human DKK-1 Protein (SinoBiological/10170-H08H) was incubated with 1.5E4 cells/well of the TCF LEF Reporter HEK-293 Cell Line (Cat. GM-C29875) in a 96-well plate for 1 hour in assay buffer (DMEM+1% FBS+1% P.S). Subsequently, the recombinant human Wnt-3a (R&D/5036-WN-CF) at a concentration of 15 ng/well was added, and the coculture proceeded for an additional 16 hours. Firefly luciferase activity was then measured using the GMOne-Step Luciferase Reporter Gene Assay Kit (Cat. GM-040503). The results indicated maximum blocking folds of approximately [52.3]. Data are shown by drug mass concentration.
The passage stability of response to Recombinant Human Wnt-3a Protein. The passage 5 and 27 of TCF LEF Reporter HEK-293 Cell Line (Cat. GM-C29875) at a concentration of 1.5E4 cells/well (96-well format) were stimulated with serial dilutions of Recombinant Human Wnt-3a Protein (R&D SYSTEMS/5036-WN/CF) in assay buffer (DMEM + 1% FBS + 1% P.S) for 16 hours. The firefly luciferase activity was measured using the GMOne-Step Luciferase Reporter Gene Assay Kit (Cat. GM-040503). Data are shown by drug mass concentration.
Specifications
Cat. NoGM-C29875
ProductTCF LEF Reporter HEK-293 Cell Line
Product Format1 vial of frozen cells
Quantity5E6 Cells per vial,1 mL
Storage ConditionsLiquid nitrogen immediately upon receipt
Recovery MediumDMEM+10% FBS+1% P.S
Growth mediumDMEM+10% FBS+1% P.S+4 μg/mL Blasticidin
NoteNone
Freezing Medium90% FBS+10% DMSO
Growth propertiesAdherent
Growth Conditions37°C, 5% CO₂
Safety considerationsBiosafety Level 2
Mycoplasma TestingThe cell line has been screened to confirm the absence of Mycoplasma species.
Materials
ReagentOrdering Information
DMEMGibco/C11995500BT
Fetal Bovine SerumCegrogen biotech/A0500-3010
Pen/StrepThermo/15140-122
BlasticidinGenomeditech/GM-040404
Recombinant Human Wnt-3a ProteinR&D SYSTEMS/5036-WN
Recombinant Human DKK-1 Protein (His Tag), HPLC-verifiedSino Biological/10170-H08H
GMOne-Step Luciferase Reporter Gene Assay KitGenomeditech/GM-040503
Cell Culture

Cell Recovery

Recovery Medium: DMEM+10% FBS+1% P.S

To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C. Storage at -70°C will result in loss of viability.

a)       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 - 3 minutes).

b)       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 of the operations from this point on should be carried out under strict aseptic conditions.

c)       Transfer the vial contents to a centrifuge tube containing 5.0 mL complete culture medium and spin at approximately 176 x g for 5 minutes. Discard supernatant.

d)       Resuspend cell pellet with the recommended recovery medium. And dispense into appropriate culture dishes.

e)       Incubate the culture at 37°C in a suitable incubator. A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.

Cell Freezing

Freezing Medium: 90% FBS+10% DMSO

a)          Centrifuge at 176 x g for 3 minutes to collect cells.

b)         Resuspend the cells in pre-cooled freezing medium and adjust the cell density to 5E6 cells/mL.

c)          Aliquot 1 mL into each vial.

d)         Place the vial in a controlled-rate freezing container and store at -80°C for at least 1 day, then transfer to liquid nitrogen as soon as possible.

Cell passage

Growth medium: DMEM+10% FBS+1% P.S+4 μg/mL Blasticidin

For the first 1 to 2 passages post-resuscitation, use the recovery medium. Once the cells have stabilized, switch to a growth medium.

a)          Subculturing is necessary when the cell density reaches 80%. It is recommended to perform subculturing at a ratio of 1:3 to 1:4 every 2-3 days. Ensure that the density does not exceed 80%, as overcrowding can lead to reduced viability due to compression.

b)         Remove and discard culture medium.

c)          Briefly rinse the cell layer with PBS to remove all traces of serum that contains trypsin inhibitor.

d)         Add 1.0 mL of 0.25% (w/v) Trypsin-EDTA solution to dish and observe cells under an inverted microscope until cell layer is dispersed (usually within 30 to 60 seconds at 37°C).

e)          Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.

f)          Add 2.0 mL of growth medium to mix well and aspirate cells by gently pipetting.

g)         After centrifugation, resuspend the pellet and add appropriate aliquots of the cell suspension to new culture vessels.

h)         Incubate cultures at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:3 - 1:4 is recommended

Medium Renewal: Every 2 to 3 days

Notes

a)          Upon initial thawing, a higher number of dead cells is observed, which is a normal phenomenon. Significant improvement is seen after adaptation. Once the cells reach a stable state, the number of dead cells decreases after subculturing and the cell growth rate becomes stable.

b)         Ensure that the cell density does not exceed 80%, as overcrowding may lead to reduced viability due to compression.

 


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