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2 protocols using nih3t3

1

Cell Culture and Maintenance Protocol

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HEK293T and HeLa cells were purchased from the JCRB Cell Bank. Lenti-X 293 T cells were purchased from Takara Bio (Shiga, Japan). NIH3T3, BEAS2B, A549, and HCC827 cells were purchased from American Tissue Culture Collection (ATCC). p53-knockout mouse embryonic fibroblasts (p53−/− MEFs) were obtained as described elsewhere73 (link). Mycoplasma contamination was routinely monitored using MycoStrip (InvivoGen, San Diego, CA, USA). HEK293T, HeLa, NIH3T3, and p53−/− MEFs were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS; Nichirei Biosciences, Tokyo, Japan). Lenti-X 293 T cells were cultured in DMEM (high glucose) supplemented with 10% FBS. BEAS2B cells were cultured in DMEM/F-12K (Nacalai Tesque) supplemented with 10% FBS. A549 and HCC827 cells were cultured in RPMI-1640 medium (Nacalai Tesque) supplemented with 10% FBS. Streptomycin (100 U/ml)/Penicillin-G (100 μg/ml) solution (Nacalai Tesque) was added to all cell culture media.
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2

Cell Line Cultivation Protocol

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The human lung adenocarcinoma cell line HCC827 was obtained from the RIKEN BioResource Center (Tsukuba, Japan) and the NCI‐H292 and NCI‐H1838 cell lines were from the ATCC (Manassas, VA, USA). The mouse fibroblast cell line NIH3T3 was obtained from the ATCC. The human embryonic kidney cell line 293FT was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Plat‐A retroviral packaging cell line was a kind gift from Dr Toshio Kitamura (Institute of Medical Science, University of Tokyo). HCC827, NCI‐H292 and NCI‐H1838 cells were cultured in RPMI 1640 (Nacalai Tesque, kyoto, Japan) supplemented with 10% FBS (Biowest, Nuaillé, France), 100 units/mL penicillin and 100 μg/mL streptomycin (Sigma‐Aldrich, St. Louis, MO, USA). NIH3T3 and 293FT cells were cultured in DMEM (Nacalai Tesque) supplemented with 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin. Plat‐A cells were cultured in DMEM with 4.5 g/L glucose supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, 1 μg/mL puromycin (Wako Pure Chemical Industries, Tokyo, Japan) and 10 μg/mL blasticidin (Kaken Pharmaceutical, Tokyo, Japan). Cells were maintained at 37°C in a humidified 5% CO2 incubator (Panasonic, Osaka, Japan).
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