Jurkat cells (ATCC, No. TIB-152) and T1 (174 x CEM.T1) (ATCC CRL-1991) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were grown at 37 °C in a humidified 95% air/5% CO2 atmosphere. Jurkat cells were cultured in RPMI 1640 medium (ATCC, No. 30-2001), and CRL-1991 cells were cultured in Iscove’s modified Dubelcco´s medium, both mediums containing 10% fetal bovine serum (FBS, ATCC, No. 30-2020) and antibiotics (penicillin and streptomycin sulphate).
Jurkat cell
Manufactured by American Type Culture Collection
Sourced in United States, United Kingdom, Germany
Jurkat cells are a human T lymphocyte cell line derived from the peripheral blood of a patient with T cell leukemia. They are widely used in research for their ability to rapidly proliferate and express T cell markers.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (23 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (10 mentions)
Rpmi 1640, by Thermo Fisher Scientific (10 mentions)
Hek293t, by American Type Culture Collection (7 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Hela cell, by American Type Culture Collection (6 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
Jurkat cells, by Thermo Fisher Scientific (5 mentions)
Rpmi 1640 medium, by American Type Culture Collection (4 mentions)
Rpmi 1640, by Merck Group (4 mentions)
Hek293t cell, by American Type Culture Collection (4 mentions)
Hek293t, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Mcf 7, by American Type Culture Collection (4 mentions)
Rpmi 1640, by American Type Culture Collection (3 mentions)
K562 cell, by American Type Culture Collection (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Merck Group (3 mentions)
L glutamine, by Merck Group (3 mentions)
116 protocols using jurkat cell
1
Culturing Jurkat and T1 Cells
2
Generation of HVEM-deficient Jurkat Cells
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Jurkat cells (ATCC) were cotransfected with the HVEM CRISPR/Cas9 KO plasmid (Santa Cruz) and the HVEM HDR plasmid (Santa Cruz) according to the manufacturer's instructions. After 48 h, all transfected cells were selected with media containing 2 μg/ml puromycin (TOCRIS Bioscience) for at least 2 weeks. Before use, the reduction of HVEM expression in the selected cells was confirmed by flow cytometry. The cells expressing lower levels of HVEM were named HVEMlow Jurkat cells. Jurkat cell lines were maintained in RPMI 1640 supplemented with 10% inactivated FBS, 100 IU penicillin plus 100 μg/ml streptomycin, and 1 mM sodium pyruvate. All cells were incubated in a humidified CO2 incubator.
3
Engineered aAPCs for T-cell Activation
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HEK293T cells (ATCC, Manassas, VA, USA), HEK293T-based aAPCs (HLA null, aAPC, and aAPC-CMV pp65), and Jurkat cells (ATCC, USA) were cultured in RPMI-1640 medium (Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (Lonza, Walkersville, MD, USA), 1% penicillin–streptomycin (Lonza, Walkersville, MD, USA), and 1% L-glutamine (Lonza, Walkersville, MD, USA). HEK293T-based aAPCs have been previously shown to stably express single HLA class I A*02:01 and co-stimulatory molecules such as CD80, CD83, CD137L, and CD32 [16 (link)]. All cells were grown and assayed at 37 °C with 5% atmospheric CO2.
4
Cell Line Maintenance and Characterization
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HEK293T and Jurkat cells were purchased from ATCC (Manassas, VA). LHL2/3 and LEL6 cells were constructed in our laboratory and used elsewhere (Jiang et al., 2021 ). LHL2/3 cells were HL2/3 cells (obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HL2/3 from Dr. Barbara K. Felber and Dr. George N. Pavlakis) encoded to highly express PD-L1 and the firefly luciferase gene. LEL6 cells are Jurkat cells that highly express Env, PD-L1, and the firefly luciferase gene. ACH-2 cells are HIV-1 latently infected CEM cells that contain a single copy of proviral DNA per cell (obtained from the NIH AIDS Reagent Program). HEK293T and LHL2/3 cells were maintained in DMEM (Gibco, Grand Island, NY) supplemented with 10% foetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin (Gibco). Jurkat and LEL6 and ACH-2 cells were maintained in RPMI1640 medium (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco).
5
Cell Line Cultivation and Characterization
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VCR-R CEM and CCRF-CEM cells were obtained from Prof. Maria Kavallaris (University of New South Wales, Sydney, Australia) [21 (link)] and were cultured in RPMI 1640 (PAN Biotech, Aidenbach, Germany) containing 10% FCS (PAN Biotech). VCR-R CEM cells are a multidrug-resistant subline of CCRF-CEM cells [22 (link)], generated by long-term exposure to increasing concentrations of vincristine [23 (link)]. Jurkat cells were bought from ATCC and cultured in RPMI 1640 containing 10% FCS and pyruvate. Cell lines were typically passaged twice to thrice a week. HeLa cells were purchased from DSMZ (Braunschweig, Germany) and cultured in DMEM (PAN Biotech) containing 10% FCS. The model of ALL patients’ leukemia cells growing in mice has been described previously [24 (link)]. In the present study, patient-derived xenograft (PDX) cells were engrafted and freshly isolated from the bone marrow or spleen of NSG mice (The Jackson Laboratory, Bar Harbour, ME, USA) and subsequently cultured in RPMI 1640 supplemented with 20% FCS and P/S. Peripheral blood mononuclear cells (PBMC) were obtained from ATCC and cultured in RPMI 1640 supplemented with 20% FCS and P/S.
Cell line STR profiling was performed. None of the cell lines used are listed in the database of commonly misidentified cell lines maintained by ICLAC. All cells are proven to be mycoplasma-free quarterly.
Cell line STR profiling was performed. None of the cell lines used are listed in the database of commonly misidentified cell lines maintained by ICLAC. All cells are proven to be mycoplasma-free quarterly.
6
Radiation Effects on Immune Cells
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Jurkat cells (Clone E6–1, ATCC, VA, USA) were irradiated with doses of 0, 1, 3, or 6Gy (Xrad 320, Precision X-ray Inc., CT, USA) and left to recover for 24 h. At days 1, 14, and 28, post-irradiation cells were mitogen-stimulated and analyzed by flow cytometry as described above. Adipocytes (3 T3-L1, CL-173, ATCC) were irradiated with doses of 0, 1, 3, and 6Gy and left to recover for 24 h prior to the collection of culture media. PBMCs from six young healthy donors were mitogen-stimulated in conditioned media from irradiated adipocytes and analyzed by flow cytometry as described above.
7
Cell Culture Protocols for Cancer Cell Lines
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Jurkat cells (ATCC CRL-2901) were grown in RPMI-1640 medium containing 10% fetal bovine serum at 37 °C and 5% CO2. K562 cells (ATCC CCL 423) were grown in IMDM medium containing 10% fetal bovine serum at 37 °C and 5% CO2, and HL-60 cells (ATCC CCL-240) were grown in IMDM medium containing 20% fetal bovine serum at 37 °C and 5% CO2.
8
Cell Line Identification and Mycoplasma Testing
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HEK-293T cells and Jurkat cells were obtained from ATCC. Identity testing was carried out by PCR. Cell lines tested negative for mycoplasma.
9
Cytotoxicity Assays Using Cell Lines
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PC12 (CRL-1721), SH-SY5Y (CRL-2266) and Jurkat cells (CRL-1990) were purchased from ATCC. PC12, Jurkat, and SH-SY5Y cells were grown at 37 °C (5% CO2 atmosphere) in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum, 5% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL of penicillin, and 100 mg/mL of streptomycin. Cell culture medium was changed three times per week, and when confluent, cells were split 1:6. For the experiments reported here, subconfluent cells were treated with different concentrations of 100–10,000 μM hydrogen peroxide, 10 μM staurosporine, or 500 μg/mL of actinomycin D.
10
Cytotoxicity Assay of Leukemia Cell Lines
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Human acute myeloid leukemia (AML) K562 cells and human acute lymphoblastic leukemia (ALL) Jurkat cells (ATCC, Manassas, USA) were maintained in Roswell Park Memorial Institute Medium (RPMI; GIBCO, São Paulo, Brazil) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 10 mm HEPES, pH 7.4. The cells were maintained in plastic culture flasks at 37o at a 5% CO2 humidified atmosphere. Cells were seeded at a density of 106 cells per bottle and medium was changed every three days. Cell viability was assessed by MTT method (3-(4,5-dimethiazol-zyl)-2-5-diphenyltetrazolium bromide, Sigma Chemical Co., St Louis, USA)[15 (link)]. Briefly, the compound was added to K562 and Jurkat cells at different concentrations (1-100 µM) and incubated for 24, 48 or 72 h. The same volume of DMSO was added to control wells. MTT solution was added to each well (10% v/v) and the plates were incubated for 3 h. After incubation, the supernatant was discarded, the formazan precipitated was dissolved with 100 ml of an isopropyl alcohol-HCl 0.04 N solution and the absorbance was determined at 540 nm. All assays were performed in triplicate and the IC50 values (50% inhibitory concentration) were calculated using GraphPad Prism software package for Windows (v. 5.0 GraphPad Prism Software Inc, San Diego, CA).
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