GAS Reporter HCT116 Cell Line

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Current position：Product Center > Cell lines > Cytokines > IFN-γ > GAS Reporter HCT116 Cell Line

GAS Reporter HCT116 Cell Line

Description

Cat. No: GM-C38096
Product: GAS Reporter HCT116 Cell Line

GAS (Gamma-Activated Sequence) is a key cis-regulatory DNA element in cytokine signaling pathways, regulating gene expression. It is a binding site for transcription factors like STATs, activated by cytokine stimuli. Discovered in interferon (IFN)-induced genes and closely linked with STAT1, GAS is commonly found in promoter regions of genes responsive to IFN stimulation. It is crucial in immunity, proliferation, and differentiation.

The GAS pathway is driven by the JAK-STAT pathway. Cytokines like IFN-γ activate Janus kinases (JAKs), which phosphorylate receptors to recruit STAT proteins. Phosphorylated STATs dimerize, move to the nucleus, and bind GAS sequences to trigger gene expression. For example, IFN-γ activates STAT1 to regulate host defense genes. This pathway plays critical roles in immunity, inflammation, and cellular growth, with abnormalities linked to autoimmune diseases, inflammation, and tumors.

GAS Reporter HCT116 Cell Line is a clonal stable HCT116 cell line constructed using lentiviral technology, along with GAS signal-dependent expression of a luciferase reporter gene. When IFNγ binds to IFNγR, it activates downstream signaling pathways, leading to the expression of luciferase. The luciferase activity measurement indicates the activation level of the signaling pathway and can thus be used to evaluate the in research of drugs related to GAS.

Data

Response to IFN-gamma and Recombinant Human IFN-α2. The GAS Reporter HCT116 Cell Line (Cat. GM-C38096) at a concentration of 2E4 cells/well (96-well format) was stimulated with serial dilutions of IFN-gamma (MCE/HY-P70610) and Recombinant Human IFN-α2 (Biolegend/592702) in assay buffer (McCoy’s 5A + 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 [58.6] and [7.4]. Data are shown by drug mass concentration.

Specifications

Cat. No		GM-C38096
Product		GAS Reporter HCT116 Cell Line
Product Format		1 vial of frozen cells
Quantity		5E6 Cells per vial,1 mL
Storage Conditions		Liquid nitrogen immediately upon receipt
Recovery Medium		McCoy's 5A+10% FBS
Growth medium		McCoy's 5A+10% FBS+1% P.S+10 μg/mL Blasticidin
Note		None
Freezing Medium		90% FBS+10% DMSO
Growth properties		Adherent
Growth Conditions		37°C, 5% CO₂
Safety considerations		Biosafety Level 2
Mycoplasma Testing		The cell line has been screened to confirm the absence of Mycoplasma species.

Materials

Reagent		Ordering Information
McCoy's 5A		VivaCell/C3020-0500
Fetal Bovine Serum		Cegrogen biotech/A0500-3010
Pen/Strep		Thermo/15140-122
Blasticidin		Genomeditech/GM-040404
IFN-gamma		MCE/HY-P70610
Recombinant Human IFN-α2 (carrier-free)		BioLegend/592702
GMOne-Step Luciferase Reporter Gene Assay Kit		Genomeditech/GM-040503

Cell Culture

Cell Recovery

Recovery Medium: McCoy's 5A+10% FBS

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: McCoy's 5A+10% FBS+1% P.S+10 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) Remove and discard culture medium.

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

c) 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 1 to 2 minutes at 37°C).

d) 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.

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

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

g) Incubate cultures at 37°C.

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

Medium Renewal: Every 1 to 2 days

Notes

a) After the stabilization of the cell condition, there will be fewer dead cells post-passage, the cell growth rate will tend to stabilize, cell morphology will become uniform, and the cells will appear robust.

Cell Recovery

Recovery Medium: McCoy's 5A+10% FBS

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: McCoy's 5A+10% FBS+1% P.S+10 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) Remove and discard culture medium.

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

c) 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 1 to 2 minutes at 37°C).

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

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

g) Incubate cultures at 37°C.

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

Medium Renewal: Every 1 to 2 days

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