Mycoplasma test kit

The human chronic myelogenous leukemia cell line K562 and subclones of the human embryonic kidney cell lines HEK293, HEK293T, and the Lenti-X 293 T cell line were obtained from JCRB Cell Bank (Osaka, Japan), RIKEN Bio-Resource Center, and Clontech (Mountain View, CA, USA), respectively. The K562/HER2, K562/HER2/scFv, and K562/HER2/scFv^B cell lines were established through lentiviral transduction of K562 and K562/HER2 cells with the plasmids pCSII/HER2-mCherry, pCSII/scFv-sfGFP, and pCSII/scFv-BFP, respectively.
K562, K562/HER2, K562/HER2/scFv, and K562/HER2/scFv^B cells, as well as HEK293T and Lenti-X 293T cells, were maintained in 10% FBS-RPMI-1640 (Invitrogen, Waltham, MA, USA) and 10% FBS-DMEM (Nacalai Tesque, Kyoto, Japan), respectively. All media were supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL) (Nacalai Tesque). Cells were maintained in a 37 °C incubator with 5% CO₂ and regularly checked for mycoplasma contamination using a mycoplasma test kit (Lonza, Basel, Switzerland).

CAD (purchaseable from Sigma, 08100805) cells were grown in media containing Ham’s F12 and DMEM (1:1) (GIBCO, Grand Island, NY) and 10% (w/v) fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) and 1% (v/v) penicillin/streptomycin (v/v), in 5% CO₂ and 95% humidified atmosphere at 37 °C. Cells were transiently transfected 48 h prior to experiments in a 1 µg:3 µl Lipofectamine 2000 (Invitrogen) ratio according to manufacturer’s protocol. The cells were regularly tested for mycoplasma with a standard mycoplasma test kit (Lonza, NJ, USA).

The Lenti-X 293 T cell line, which is a subclone of the human embryonic kidney cell line HEK 293, human cervical cancer cell line HeLa, and human chronic myelogenous leukemia cell line K562 were obtained from Clontech (Mountain View, CA, USA), RIKEN Bio-Resource Center, and JCRB Cell Bank (Osaka, Japan), respectively. K562/CD25 and K562/CD25/scFv cell lines were established by lentiviral transduction of K562 and K562/CD25 cells with pCSII/CD25-mRuby2 and pCSII/scFv-sfGFP vectors, respectively, followed by cell cloning through serial dilution.
Lenti-X 293 T cells, and K562, K562/CD25, and K562/CD25/scFv cells were maintained at 37 °C with 5% CO₂ in 10% FBS-DMEM (Nacalai Tesque, Kyoto, Japan) and 10% FBS-RPMI-1640 (Invitrogen, Carlsbad, CA, USA), respectively. All media were supplemented with penicillin (100 U/mL) and streptomycin (100 µg/mL) (Nacalai Tesque). Cells were regularly checked for mycoplasma contamination using a mycoplasma test kit (Lonza, Basel, Switzerland).