J rt3 t3

Manufactured by American Type Culture Collection

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The J.RT3-T3.5 is a laboratory equipment product designed for specific applications. It serves a core function without further interpretation or extrapolation on its intended use.

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7 protocols using j rt3 t3

Characterization of Immune Cell Lines

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J.RT3-T3.5 and Jurkat (Clone E6–1) cell lines used for counter selection and nucleofection, respectively, were purchased from ATCC. The Epstein-Barr virus transformed lymphoblastoid cell line (TM-LCL) used in the rapid expansion protocol (REP) of T cells was made from mononuclear cells as previously described.⁵⁷ CD19⁺ and OKT3⁺ K562 cells used for functional assays were generated by lentivirally transducing parental K562 parental cells (ATCC) with CD19- or OKT3-expressing constructs. Raji parental cells were also purchased from ATCC. All the above cell lines were cultured in RPMI 1640 medium (Gibco) with 10% heat-inactivated FBS (Life Tech and VWR). Human peripheral blood mononuclear cells (PBMCs) were isolated from Leukocyte Reduction System (LRS) cones (Bloodworks Northwest) using Ficoll-Paque density gradient centrifugation (GE). Mixed or CD8⁺ T cells used in non-isolation experiments were a generous gift from Juno Therapeutics. Rhesus PBMCs were a generous gift from Dr. Agne Taraseviciute (Seattle Children’s Research Institute).

Kacherovsky N., Cardle I.I., Cheng E.L., Yu J.L., Baldwin M.L., Salipante S.J., Jensen M.C, & Pun S.H. (2019). Traceless aptamer-mediated isolation of CD8+ T cells for CAR-T cell therapy. Nature biomedical engineering, 3(10), 783-795.

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Generation of HEK-293T-I-Ag7 Cell Line

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The HEK-293T-I-A^g7 cell line was generated by sequential transduction of HEK-293T (ATCC, #CRL-11268) cells with lentivirus encoding for I-

A_{α}^{d} / C F P

, I-

A_{β}^{g 7} / G F P

, HLA-DM_α/β/GFP, and human CD74 (invariant chain -Ii-) fused to human CD80 (hCD80) with sequential flow cytometry-based cell-sorting guided by CFP fluorescence (reporting I-

A_{α}^{d}

expression), GFP (reporting DM_α/β expression), anti-hCD80 (reporting Ii, expression), and anti-I-A^g7 (reporting I-

A_{α}^{d}

/I-

A_{β}^{g 7}

heterodimer expression).
4.1- and BDC2.5-JurMA cells [derived from the TCR_β-null Jurkat cell line J.RT3-T3.5 (ATCC) and carrying a NFAT-driven luciferase reporter] were generated by transducing JurMA cells with retroviruses encoding monocistronic, P2A-tethered TCR_α-TCR_β chains.

Parras D., Solé P., Delong T., Santamaría P, & Serra P. (2021). Recognition of Multiple Hybrid Insulin Peptides by a Single Highly Diabetogenic T-Cell Receptor. Frontiers in Immunology, 12, 737428.

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Cell Culture Conditions for Cell Lines

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SW620 (ATCC), Lenti-X 293T (Takara Bio), and A375 (ATCC) cell lines were cultured in DMEM media containing 10% FBS, penicillin, and streptomycin. NCI-H441 (ATCC) and Jurkat that has the TCR beta-chain-deficient mutant (J.RT3-T3.5, ATCC) were cultured in RPMI-1640 containing 10% FBS, penicillin, and streptomycin.

Choi J., Goulding S.P., Conn B.P., McGann C.D., Dietze J.L., Kohler J., Lenkala D., Boudot A., Rothenberg D.A., Turcott P.J., Srouji J.R., Foley K.C., Rooney M.S., van Buuren M.M., Gaynor R.B., Abelin J.G., Addona T.A, & Juneja V.R. (2021). Systematic discovery and validation of T cell targets directed against oncogenic KRAS mutations. Cell Reports Methods, 1(5), 100084.

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Characterization of Immune Cell Lines

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Establishing Jurkat-CD8-NFAT Reporter Cell Line

Cited 1 time

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Cell lines were cultured in Roswell Park Memorial Institute (RPMI)-1640 (T2 and J.RT3-T3.5), or DMEM (293T, CaSki, and SCC90) or MEM (SK-MEL-28), supplemented with 10% Fetal Bovine Serum (Lonsera) and 1% penicillin/streptomycin (Life Technologies). T2, 293T, SK-MEL-28, and J.RT3-T3.5 cell lines were purchased from American Type Culture Collection (ATCC)CaSki and SCC90 cell lines were purchased from CTCC. CaSki is an HPV16⁺ cervical cancer cell line. SCC90 is an HPV16⁺ head and neck squamous cell carcinoma cell line. All cell lines used in the experiments were mycoplasma-free.
HPV16 E6, HPV16 E7, and HLA-A*11:01 heavy chain genes were synthesized and constructed into pLKO.1puro (addgene 8453) lentiviral vectors under EF-1α promoter. P2A-linked GFP was used as the reporter gene.
The J.RT3-T3.5 cell line was used to generate Jurkat-CD8-NFAT (JK8NF) reporter cell line. First, J.RT3-T3.5 cells were transduced with lentiviral particles carrying the pLenti-human CD8 (hCD8) transfer plasmid and sorted by FACS based on high CD8 expression (Jurkat-CD8 cells). Next, the Jurkat-CD8 cells were transduced with lentiviruses encoding the nuclear factor of activated T cells (NFAT)-inducible ZsGreen reporter genes.29 (link) Finally, JK8NF clones were selected based on a strong ZsGreen signal following Phorbol-12-myristate-13-acetate (PMA)/ionomycin stimulation.

Xiong C., Huang L., Kou H., Wang C., Zeng X., Sun H., Liu S., Wu B., Li J., Wang X., Wang Z, & Chen L. (2022). Identification of novel HLA-A*11:01-restricted HPV16 E6/E7 epitopes and T-cell receptors for HPV-related cancer immunotherapy. Journal for Immunotherapy of Cancer, 10(9), e004790.

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Latent HIV-1 Model Cell Lines

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J1.1 (derived from Jurkat) [43 (link)] and ACH-2 (derived from A3.01) cells [44 (link)], which harbor the latent HIV-1 LAV strain and their parental Jurkat and A3.01 cells, Jβ2.7 cells (LFA-1α-deficient Jurkat cells) [45 (link)], and J.RT3-T3.5 (TCR β chain-deficient Jurkat cells) (American Type Culture Collection, Manassas, VA, USA) were grown in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS). HS-5 and HS-27A cells (human stromal cell lines derived from bone marrow) (American Type Culture Collection) were grown in Dulbecco’s modified Eagle’s medium and RPMI1640 medium, supplemented with 10% FBS.

Okutomi T., Minakawa S., Hirota R., Katagiri K, & Morikawa Y. (2020). HIV Reactivation in Latently Infected Cells with Virological Synapse-Like Cell Contact. Viruses, 12(4), 417.

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Isolation of Human CD4+ T Cells

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Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood using a Ficoll-Paque PLUS (GE Amersham) gradient. CD4⁺ T cells were isolated using magnetic beads conjugated with the anti-CD4 Ab and a magnetic column (Miltenyi Biotec). Jurkat cells [10] (link) and J.RT3-T3.5 [11] (link), [12] (link) were purchased from the American Type Culture Collection (ATCC).

Shiheido H., Chen C., Hikida M., Watanabe T, & Shimizu J. (2014). Modulation of the Human T Cell Response by a Novel Non-Mitogenic Anti-CD3 Antibody. PLoS ONE, 9(4), e94324.

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