Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+)

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CJUR-STK269-2Vials (1Vial X 2)

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Product Details
Performance Data
Reviews & Citations
Important Statement
Resources

1. Genetically modified cell lines best reflect MOA (Mechanism of Action)
2. Higher activity and larger assay window for robust and reproducible cell-based bioassay
3. Comprehensive application data to support assay development and validation
4. Full tracible record, stringent quality control and validated cell passage stability
5. Parental cell line legally obtained from internationally recognized cell resource bank and commercially licensed
6. Global commercial license assistance whenever regulatory filing is required
Description
The Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+) was engineered to not only express the NFAT signaling response element and the full length human CD8A (Uniprot: P01732) and CD8B (Uniprot: P10966), but also targeted knockout of human TRAC(Gene ID: 28755), TRBC1 (Gene ID: 28639)，TRBC2 (Gene ID: 28638) and CD4 (Gene ID: 920), The expression level of human TCRαβ，CD4 and CD8 were confirmed by flow cytometry. Mutated sequences of human TCRαβ and CD4 produced by nonhomologous end joining (NHEJ) were confirmed through genomic sequencing. This reporter cell can be equipped with either a chimeric antigen receptor (CAR) or a T cell receptor (TCR), providing a cell-based functional platform for CAR or TCR screening and characterization by detecting the change in luminescence.
Application
• Screen for CAR.
• Screen for MHC I restricted antigen-specific TCR.
Growth Properties
Suspension
Selection Marker
Puromycin (5 μg/mL) + Hygromycin B (20 μg/mL)
Complete Growth Medium
RPMI-1640 + 10% FBS
Freeze Medium
Serum-free cell cryopreservation medium
Quantity
1 vial contains at least 5×10^6 cells in 1 mL serum-free cryopreservation medium
Storage
Frozen in liquid nitrogen.
Mycoplasma Testing
Negative
Sterility Testing
Negative
Instructions for Use
See data sheet for detailed culturing and assay protocol.
ACRO Quality Management System

Receptor Assay
Expression analysis of human TCRαβ on Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+) by FACS.
Cell surface staining was performed on Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+) using PE-labeled anti-human TCRαβ antibody. The parental NFAT (Luc) Jurkat Reporter Cell (Cat. No. SCJUR-STF046) was stained with PE-labeled anti-human TCRαβ antibody as the positive control cell. The parental NFAT (Luc) Jurkat Reporter Cell was stained with PE-labeled isotype control antibody as the negative control cell.
Protocol
Expression analysis of human CD3 on Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+) by FACS.
Cell surface staining was performed on Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+) using APC-labeled anti-human CD3 antibody. The parental NFAT (Luc) Jurkat Reporter Cell (Cat. No. SCJUR-STF046) was stained with APC-labeled anti-human CD3 antibody as the positive control cell. The parental NFAT (Luc) Jurkat Reporter Cell was stained with APC-labeled isotype control antibody as the negative control cell.
Protocol
Expression analysis of human CD4 and human CD8 on Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+) by FACS.
Cell surface staining was performed on Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+) using PE-labeled anti-human CD4 antibody and APC-labeled anti-human CD8 antibody. The Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+) was stained with PE-labeled isotype control antibody and APC-labeled isotype control antibody as the negative control cell.
Protocol

Signaling Bioassay
Response to anti-human CD3 antibody (RLU).
Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+) and the parental NFAT (Luc) Jurkat Reporter Cell (Cat. No. SCJUR-STF046) were incubated with serial dilutions of anti-human CD3 antibody (Cat. No. CDE-M120a). Compared to the parental NFAT (Luc) Jurkat Reporter Cell, Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+) exhibited no activation response to anti-human CD3 antibody stimulation.
Protocol
Response to SEE (RLU).
Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+) and the parental NFAT (Luc) Jurkat Reporter Cell (Cat. No. SCJUR-STF046) were incubated with serial dilutions of SEE in the presence of target cells expressing MHC II. Compared to the parental NFAT (Luc) Jurkat Reporter Cell, Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+) exhibited no activation response to SEE stimulation in the presence of target cells expressing MHC II.
Protocol
Response to PMA plus Ionomycin (RLU).
Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+) was stimulated with serial dilutions of PMA plus Ionomycin (1 μM). The EC50 was approximately 0.23ng/mL.
Protocol
Response to PMA plus Ionomycin (FOLD).
Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+) was stimulated with serial dilutions of PMA plus Ionomycin (1 μM). The max induction fold was approximately 882.
Protocol

Sequencing Analysis
Genomic Sequencing of human TRAC in the Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+).
Sanger sequencing was used for mutation analysis of human TRAC. The sequencing results demonstrated that frameshift mutations were generated in the human TRAC gene in the Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+).
Genomic Sequencing of human TRBC1 in the Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+).
Sanger sequencing was used for mutation analysis of human TRBC1. The sequencing results demonstrated that frameshift mutations were generated in the human TRBC1 gene in the Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+).
Genomic Sequencing of human TRBC2 in the Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+).
Sanger sequencing was used for mutation analysis of human TRBC2. The sequencing results demonstrated that frameshift mutations were generated in the human TRBC2 gene in the Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+).
Genomic Sequencing of human CD4 in the Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+).
Sanger sequencing was used for mutation analysis of human CD4. The sequencing results demonstrated that frameshift mutations were generated in the human CD4 gene in the Human TCR Knockout (Luc) Jurkat Reporter Cell (CD8+).