H_PD-1 Reporter Jurkat Cell Line

Cat. No: GM-C07928
Product: H_PD-1 Reporter Jurkat Cell Line

PD-1 is an immunosuppressive receptor on activated T and B cells, essential for regulating immune responses to tumor antibodies and self-antigens. Its interaction with ligands PD-L1 or PD-L2 inhibits TCR signaling, affecting cell proliferation, transcriptional activation, and cytokine production. Therapeutic antibodies and Fc fusion proteins blocking this interaction have shown promise in cancer clinical trials.

H_PD-1 Reporter Jurkat Cell Line is a stable clonal Jurkat cell line constructed using lentiviral technology, constitutive expression of the PD-1 gene, along with signal-dependent expression of a luciferase reporter gene. It has three applicable cells: when co-cultured with the aAPC(OKT3) PDL1 CHO-K1 cell line or the H_PD-L1 Raji cell line, the PD-1/PD-L1 interaction inhibits TCR signaling and luciferase expression. Adding antibodies that block this interaction can relieve the inhibition, restoring TCR signaling and luciferase expression. When co-cultured with the H_CD32B aAPC CHO-K1 cell line, the addition of PD-1 agonist antibodies allows the antibodies to crosslink with the FcγRIIb receptor, activating PD-1 downstream SHP2-mediated inhibitory signaling, thereby suppressing downstream TCR activation signals.

Data

Response to Anti-H_CD274(PDL1) hIgG1 Antibody(Atezolizumab). Serial dilutions of the Anti-H_CD274(PDL1) hIgG1 Antibody (Atezolizumab) (Cat. GM-31740AB) was incubated with 1E4 cells/well of the aAPC(OKT3) PDL1 CHO-K1 Cell Line (Cat. GM-C05269) in a 96-well plate for 1 hour. Subsequently, H_PD-1 Reporter Jurkat Cell Line (Cat. GM-C07928) with a concentration of 1E5 cells/well was added, and the co-culture proceeded for an additional 16 hours in assay buffer (RPMI 1640 + 1% FBS + 1% P.S). Firefly luciferase activity is then measured using the GMOne-Step Luciferase Reporter Gene Assay Kit (Cat. GM-040503). The results indicated a maximum fold of approximately [5.1]. Data are shown by drug mass concentration.

Response to Anti-PD1 hIgG4 Antibody(Pembrolizumab). aAPC(OKT3) PDL1 CHO-K1 Cell Line (Cat. GM-C05269) was seeded at a density of 1E4 cells/well in a 96-well plate and incubated overnight. The next day, serial dilutions of the Anti-PD1 hIgG4 Antibody(Pembrolizumab) (Cat. GM-52674AB) ,and Human IgG1 Isotype Control were incubated with 1E5 cells/well of the H_PD-1 Reporter Jurkat Cell Line (Cat. GM-C07928) in a 96-well plate for 1 hour, and then added to the pre-seeded cells. The mixture was incubated for an additional 6 hours. Firefly luciferase activity is then measured using the GMOne-Step Luciferase Reporter Gene Assay Kit (Cat. GM-040503). The results indicated a maximum fold of approximately [7.3]. Data are shown by drug mass concentration.

Response to Anti-H_CD274(PDL1) hIgG1 Antibody (Atezolizumab). The Atezolizumab (Cat. GM-31740AB) was serially diluted and incubated with 2E4 cells/well of the H_PD-L1 Raji Cell Line in a 96-well plate for 30 minutes. Meanwhile, 1E5 cells/well of the H_PD-1 Reporter Jurkat Cell Line (Cat. GM-C07928) was pre-incubated with 0.5 ng/well of SEE in a 96-well plate for 30 minutes. Afterward, the two treated cell mixtures were combined in equal volumes and co-incubated in assay buffer (RPMI 1640 + 1% FBS + 1% P.S.) for 16 hours. Firefly luciferase activity was then measured using the GMOne-Step Luciferase Reporter Gene Assay Kit (Cat. GM-040503). The results indicated a maximum fold of approximately [8.3]. Data are presented based on drug mass concentration.

Response to Anti-PD1 hIgG4 Antibody(Pembrolizumab). The Pembrolizumab (Cat. GM-52674AB) was serially diluted and incubated with 1E5 cells/well of the H_PD-1 Reporter Jurkat Cell Line(Cat. GM-C07928) in a 96-well plate for 30 minutes. Meanwhile, 2E4 cells/well of the H_PD-L1 Raji Cell Line (Cat. GM-C03541) was pre-incubated with 0.5 ng/well of SEE in a 96-well plate for 30 minutes. Afterward, the two treated cell mixtures were combined in equal volumes and co-incubated in assay buffer for 16 hours. IgG Isotype is the control. Firefly luciferase activity was then measured using the GMOne-Step Luciferase Reporter Gene Assay Kit (Cat. GM-040503). The results indicated a maximum fold of approximately [6.6]. Data are presented based on drug mass concentration.

Response to Rosnilimab, Peresolimab and Pembrolizumab. Serial dilutions of the Rosnilimab, Peresolimab and Pembrolizumab was incubated with 1E4 cells/well of the H_CD32B aAPC CHO-K1 Cell Line (Cat. GM-C25754) in a 96-well plate for 1 hour. Subsequently, H_PD-1 Reporter Jurkat Cell Line (Cat. GM-C07928) with a concentration of 1E5 cells/well was added, and the co-culture proceeded for an additional 6 hours in assay buffer (RPMI 1640 + 1% FBS + 1% P.S). Firefly luciferase activity is then measured using the GMOne-Step Luciferase Reporter Gene Assay Kit (Cat. GM-040503). The results indicated maximum blocking folds of approximately [5.8],[5.7]and [2.1], respectively. Data are shown by drug mass concentration.

The passage stability of response to Anti-H_CD274(PDL1) hIgG1 Antibody(Atezolizumab). Serial dilutions of the Atezolizumab(Cat. GM-31740AB) were incubated with 1.5E4 cells/well of the aAPC(OKT3) PDL1 CHO-K1 Cell Line (Cat. GM-C05269) in a 96-well plate for 1 hour in assay buffer (RPMI 1640 + 1% FBS + 1% P.S). Subsequently, the passage 4, 14, and 24 of H_PD-1 Reporter Jurkat Cell Line (Cat. GM-C07928) at a concentration of 1E5 cells/well was added, and the coculture proceeded for an additional 16 hours. Firefly luciferase activity is then measured using the GMOne-Step Luciferase Reporter Gene Assay Kit (Cat.GM-040503). Data are shown by drug mass concentration.

H_PD-1 Reporter Jurkat Cell Line (Cat. GM-C07928) was determined by flow cytometry using Anti-PD1 hIgG1 Antibody (In house).

Specifications

Cat. No		GM-C07928
Product		H_PD-1 Reporter Jurkat 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		RPMI 1640+10% FBS+1% P.S
Growth medium		RPMI 1640+10% FBS+1% P.S+3.5 μg/mL Blasticidin+0.75 μg/mL Puromycin
Note		None
Freezing Medium		90% FBS+10% DMSO
Growth properties		Suspension
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
RPMI 1640		VivaCell/C3010-0500
Fetal Bovine Serum		Cegrogen biotech/A0500-3010
Pen/Strep		Thermo/15140-122
Blasticidin		Genomeditech/GM-040404
Puromycin		Genomeditech/GM-040401
aAPC(OKT3) PDL1 CHO-K1 Cell Line		Genomeditech/GM-C05269
H_PD-L1 Raji Cell Line		Genomeditech/GM-C03541
Staphylococcal Enterotoxin E (SEE) 金黄色葡萄球菌肠毒素E（SEE肠毒素）		Genomeditech/GM-H23036
Anti-H_CD274(PDL1) hIgG1 Antibody(Atezolizumab)		Genomeditech/GM-31740AB
Anti-PD1 hIgG4 Antibody(Pembrolizumab)		Genomeditech/GM-52674AB
GMOne-Step Luciferase Reporter Gene Assay Kit		Genomeditech/GM-040503

Cell Culture

Cell Recovery

Recovery Medium: RPMI 1640+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 complete medium. And dispense the suspension into 1 - 2 T-25 culture flasks.

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: RPMI 1640+10% FBS+1% P.S+3.5 μg/mL Blasticidin+0.75 μg/mL Puromycin

Approximately 48-72 hours after the initial thawing, the cells can be passaged for the first time. After this initial passage, the culture medium can be adjusted to growth medium supplemented with antibiotics. If cells are not passaged within 48 hours, it is recommended to add some fresh recovery medium and place the flask horizontally.

a) When the cell density reaches 1.5 - 2E6 cells/mL, subculture the cells. Do not allow the cell density to exceed 2E6 cells/mL.

b) It is recommended to use T-25 flasks for subculturing.

c) These cells are suspension cells, and it is recommended to use the "half-medium change" method to maintain optimal cell conditions during passaging.

d) During passaging, you can directly add fresh growth medium to the culture flask, gently pipette to resuspend the cells, and then transfer the cell suspension to a new T-25 flask for continued culture.

Subcultivation Ratio: Maintain cultures at a cell concentraion between 3E5 and 1E6 viable cells/mL.

Medium Renewal: Every 2 to 3 days

Notes

a) These cells are sensitive to density, so please ensure that the cell density is maintained within an appropriate range during culture and subculturing.

b) During the first passage, pay attention to the nutrient supply; if not subculturing, make sure to add fresh recovery medium every other day as needed.