Jurkat t cell line

HeLa cells (ATCC) were maintained in Dulbecco modified Eagle medium supplemented with 10% (vol/vol) fetal bovine serum (FBS). The Jurkat T cell line (ATCC) was maintained in RPMI 1640 supplemented with 10% (vol/vol) FBS. All media were supplemented with 2 mM l-glutamine. Plasmids used were the infectious HIV-1 molecular clone pNL4-3 (63 (link)); two derivatives with point mutations at SP1 residue 3 (SP1-A3V and SP1-A3T) (39 (link)); four derivatives with point mutations in CA at residues 156 (CA-G165E), 157 (CA-P157S), 160 (CA-P160L), and 225 (CA-G225D) (kind gift from Eric Freed, NIH, USA) (4 (link)); and two protease-defective derivatives, pNL4-3/PR⁻ with the active-site mutation PR-D25N, which contains either wild-type (WT) Gag or the SP1-A3V mutation (64 (link)). Plasmid DNA was purified with a plasmid purification maxiprep kit (Qiagen) and adjusted to 1 μg/μl. HeLa cells were transfected by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, and Jurkat T cells were transfected by using DEAE-dextran (65 (link)).

The human Jurkat T cell line was obtained from ATCC (American Type Culture Collection). The KSHV-infected BC-1 and BCBL-1 cell lines were a generous gift from Dr. Ronit Sarid (Bar-Ilan University, Israel). Cell lines were cultured in complete RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Gibco, Carlsbad, CA), and maintained at 37°C in a humidified 5% CO₂ incubator.

The EL4 mouse T cell line and the Jurkat T cell line were obtained from the American Type Culture Collection and cultured as previously described^{70 (link),71 (link)}. Briefly, cells were cultured in RPMI 1640 containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-mercaptoethanol (all from Nacalai Tesque, Kyoto, Japan) in a humidified chamber at 37 °C containing 5% CO₂.
Mouse peripheral blood and lymph node T cells were expanded from the cell suspension described above. Cells were re-suspended in RPMI-1640 containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-mercaptoethanol and incubated for 2 h in a humidified chamber at 37 °C containing 5% CO₂. Non-adherent cells were collected and incubated for another 2 h to remove monocytes. Finally, non-adherent cells were washed three times and cultured with 1 µg/ml anti-CD3 antibody (BD Pharmingen, clone 145-2C11, #553058) and 2 µg/ml anti-CD28 antibody (BD Pharmingen, clone 37.51, #557393) for 2–3 days.

Binding experiments and immunofluorescence assays were performed in the Jurkat T cell line (Clone E6-1, American Type Culture Collection, Manassas, VA,20110. USA). Jurkat cells were routinely cultured in RPMI 1640 medium, supplemented with 10% heat inactivated fetal bovine serum (FBS) and antibiotics: 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified 95% air and 5% CO2. Fresh human blood to isolate primary cells were purchased from Allcells LLC. (Alameda, CA 94502).

The human Jurkat T cell line was obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA) and maintained in RPMI 1640 medium containing 10% FBS [9 (link)].

Human Jurkat T cell line was obtained from the American Type Culture Collection (ATCC) and used to study the effect of MES on immune system. Cells were cultured in RPMI-1640 medium (Wako) supplemented with 10% inactivated fetal bovine serum (FBS), 1% antibiotics (P/S; Penicillin G (100 units/mL), Streptomycin (100 μg/mL)). Inactivation of FBS was performed in 56°C for 30 min. The culture was not supplemented with growth factors. Cells were maintained (not more than 3 x 10⁶ cells/mL) at 37°C in humidified 5% CO₂ incubator. During treatment with drugs or MES, FBS-free medium was used. For stimulation, cells were treated with 10 nM phorbol 12-myristate 13-acetate and 500 nM Ionomycin (PMA/Io), and incubated for 3 hr at 37°C. Medium was changed and total RNA was isolated.