Jurkat E 6.1 cells (Sigma Aldrich) were grown at a density of 5×105 cells/ml in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS) at 37 °C in a humidified atmosphere at 5% CO2. Every 2 days the cells were split when 80% confluence was reached. CHO cells (Sigma Aldrich) were cultured in DMEM containing 10% heat-inactivated FBS. Cells were grown in culture flasks at 37 °C in a 5% CO2-humidified incubator. Cells were then allowed to grow to 70-80% confluence before transfection. Plasmid coding β3 (human) GABAAR were transfected into CHO cells with TurboFect (ThermoFisher) transfection reagent according to the manufacturer’s protocol. Electrophysiological recordings were performed 24–48 h after transfection at room temperature.
Jurkat e 6.1 cells
Manufactured by Merck Group
Jurkat E 6.1 cells are a T lymphocyte cell line derived from a human T cell leukemia. They are commonly used in immunological and cell biology research applications.
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Lab products found in correlation
Turbofect, by Thermo Fisher Scientific (2 mentions)
Cho cells, by Merck Group (1 mentions)
Clone okt3, by Thermo Fisher Scientific (1 mentions)
Britelite, by PerkinElmer (1 mentions)
Pe conjugated anti flag antibody, by BioLegend (1 mentions)
Sureentry transduction reagent, by Qiagen (1 mentions)
Bright glo, by Promega (1 mentions)
Gene pulser, by Bio-Rad (1 mentions)
2 protocols using jurkat e 6.1 cells
1
GABA Receptor Expression in CHO Cells
2
Engineered NFAT Reporter Cell Line for CAR Signaling Assay
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A reporter cell line for Nuclear Factor of Activated T-cells (NFAT) signaling was generated from Jurkat E6.1 cells (Sigma) transduced with Cignal Lenti NFAT Reporter virus (Qiagen) using SureENTRY Transduction reagent (Qiagen). Transduced cells were selected with puromycin then single cell cloned. A clonal pathway reporter cell line, JNL10, was selected on the basis of its responsiveness to stimulation with plate-bound mouse anti-human CD3 antibody (Clone OKT3, eBioscience, RRID:AB_467057).
To assess CAR signaling activity, JNL10 reporter cells were electroporated with CAR constructs using a Bio-Rad GenePulser and cultured at 37°C overnight in a 12-well plate. The next day, electroporated JNL10 cells were activated with target cells by incubating at approximately 1:1 ratio in 100-μL culture medium in a 96-well plate for 5 hours at 37°C. NFAT-mediated signaling was monitored by the subsequent addition of Luciferase substrate (Bright-Glo, Promega or BriteLite, PerkinElmer) and measuring relative luminescence units using a plate reader. Target cells with and without electroporated JNL10 cells were used as the background control. Percentage of CAR expression on JNL10 cells was measured in parallel by staining with PE-conjugated anti-FLAG antibody (BioLegend, RRID:AB_2563147).
To assess CAR signaling activity, JNL10 reporter cells were electroporated with CAR constructs using a Bio-Rad GenePulser and cultured at 37°C overnight in a 12-well plate. The next day, electroporated JNL10 cells were activated with target cells by incubating at approximately 1:1 ratio in 100-μL culture medium in a 96-well plate for 5 hours at 37°C. NFAT-mediated signaling was monitored by the subsequent addition of Luciferase substrate (Bright-Glo, Promega or BriteLite, PerkinElmer) and measuring relative luminescence units using a plate reader. Target cells with and without electroporated JNL10 cells were used as the background control. Percentage of CAR expression on JNL10 cells was measured in parallel by staining with PE-conjugated anti-FLAG antibody (BioLegend, RRID:AB_2563147).
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