Jurkat t lymphoma clone e6 1

HEK293T (ATCC CRL-3216), HT-29 (ATCC HTB-38), J774A.1 (ATCC TIB-67) and Jurkat T lymphoma (clone E6-1, ATCC TIB-152) and L929 (ATCC CCL-1) cell lines were obtained from ATCC. Cell lines were examined for mycoplasma contamination using a Mycoplasma Detection Kit (R&D). HT-29 cells and Jurkat cells were cultured in complete RPMI-1640 medium containing 10% fetal calf serum (Invitrogen Life Technology), 10 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 50 μM 2-mercaptoethanol. HEK293T, J774A.1 and L929 cells were cultured in complete DMEM medium with the same supplements as for complete RPMI medium. Bone marrow cells were collected from tibias and femurs by flushing with cold phosphate buffered saline (PBS). Bone marrow cells were cultured in complete DMEM medium with 20% L929-conditioned media for 6 days to generate bone marrow-derived macrophages (BMDMs). Bone marrow cells were also differentiated in complete RPMI medium containing 20 ng/ml GM-CSF for 8 days to generate bone marrow-derived dendritic cells (BMDCs). DAPK was also knocked down in HT-29 cells by transfection with siDAPK1 using Lipofectamine 2000.

HEK293T (ATCC CRL-3216), HeLa (ATCC CCL-2) and Jurkat T lymphoma (clone E6-1, ATCC TIB-152) were obtained from ATCC. Cell lines were examined for mycoplasma contamination using Mycoplasma Detection Kit (R&D). Normal T cells and Jurkat cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (Invitrogen Life Technology), 10 mM glutamine, 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin, and 50 μM 2-ME (complete RPMI medium). HEK293T and HeLa cells were cultured in DMEM medium with the same supplements as in complete RPMI medium. For hypoxia (1% O₂) experiments, T cells were cultured in the cell incubator containing an oxygen controller ProOx-110 (BioSpherix) using gas of 5% CO₂ and 95% N₂ to reduce the oxygen content. Expression of DAPK or its mutants was performed by transfection of T cells with DAPK, [K42A]DAPK, or [ΔCAM]DAPK through electroporation using the MP-100 system (Life Technologies).