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4 protocols using sk ov 3 ovarian carcinoma

1

Cell Culture Protocols for Cancer and Immune Cell Lines

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MDA-MB-453, MDA-MB-468 and JIMT-1 breast carcinoma, LN-229 glioblastoma, SK-OV-3 ovarian carcinoma, HEK 293 T embryonic kidney (all ATCC, Manassas, VA) and MZ-Mel-2 melanoma cells (kindly provided by Elke Jäger, Krankenhaus Nordwest, Frankfurt, Germany) were cultured in DMEM medium (Gibco, Thermo Fisher Scientific, Darmstadt, Germany), SK-BR-3 breast carcinoma cells (ATCC) in Advanced DMEM/F12 medium (Gibco, Thermo Fisher Scientific), and K562 erythroleukemia cells (ATCC) in RPMI 1640 medium (Gibco, Thermo Fisher Scientific). All media were supplemented with 10% heat-inactivated FBS (Capricorn Scientific, Ebsdorfergrund, Germany), 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin (all Gibco, Thermo Fisher Scientific). Expi293F cells were cultured in Expi293 expression medium (both Gibco, Thermo Fisher Scientific). NK-92 cells [20 (link)] (kindly provided by NantKwest, Inc., Culver City, CA) and ErbB2-specific NK-92/5.28.z cells [21 (link), 22 (link)] were cultured in X-VIVO 10 medium (Lonza, Cologne, Germany) supplemented with 5% heat-inactivated human AB plasma (German Red Cross Blood Donation Service Baden-Württemberg-Hessen, Frankfurt, Germany) and 100 IU/mL IL-2 (Proleukin; Novartis Pharma, Nürnberg, Germany).
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Cell Culture Protocols for Cancer Research

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A-10 embryonic rat aortic smooth muscle, HeLa cervical carcinoma, SK-OV-3 ovarian carcinoma, and MDA-MB-231 breast carcinoma cell lines were obtained directly from ATCC (Manassas, VA). The MDA-MB-435 melanoma cells were acquired from the Lombardi Cancer Center (Georgetown University) and validated by ATCC. NCI/ADR-RES cells were obtained from the NIH (Bethesda, MD). The SK-OV-3/MDR-1–6/6 and HeLa wild-type βIII (WT βIII) cell lines were described previously.17 (link) MDA-MB-435, MDA-MB-231 and NCI/ADR-RES cells were cultured in Improved Minimum Essential Medium (Richter’s Modification) (Gibco, Life Technologies, Grand Island, NY) with 10% fetal bovine serum and 25 μg/mL gentamicin. WT βIII cells were grown in DMEM (Gibco, Life Technologies) with 10% fetal bovine serum and 50 μg/mL gentamicin, while all the other cell lines were maintained in Basal Medium Eagle (Sigma-Aldrich) with 10% fetal bovine serum and 50 μg/mL gentamicin. All cells were maintained in a humidified incubator at 37 °C with 5% CO2.
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Cell Line Characterization and Cultivation

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Human A549 non-small cell lung carcinoma (male), HeLa cervix carcinoma and SKOV3 ovarian carcinoma were obtained from American Type Culture Collection (ATCC). The cells were authenticated by ATCC and used within six months after thawing. Human pancreatic duct epithelial cells (H6C7) were purchased from Kerafast. Human immortalized mammary fibroblasts (HMF3) were kindly provided by Michael J. O'Hare (Ludwig Institute for Cancer Research, London, UK).61 (link) Lenti-X™ 293T human embryonic kidney epithelial cells transformed with Adenovirus type 5 DNA were from Clontech (632180). A549-triple cells (here referred to as A549-RFP-STAT3 cells) were kindly provided by Dmitry Malkov (Sigma-Aldrich, St. Louis, Missouri, USA).34 (link) Human MCF7 mammary carcinoma cells stably transfected with inducible pTRE-p95ErbB2 or corresponding pTRE vector were established and cultured as described previously.13 (link),43 (link) The expression of p95ErbB2 was induced by washing off the tetracycline (1 µg/ml) three passages prior to the experiment. H6C7 cells were cultured in SFM. Other cells were cultured in DMEM medium supplemented with 10% heat-inactivated fetal calf serum and penicillin/streptomycin (complete medium), and for H6C7cells also with 2 mM glutamine. Cells were maintained at 37°C and 5% CO2. All cell lines were found negative for mycoplasma using Venor®GeM Classic PCR kit.
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Establishment of CEA-Expressing Cell Lines

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A-431 vulvar squamous carcinoma and SK-OV-3 ovarian carcinoma cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). HER1- and HER2-expressing A-431 cells were transfected with the genes encoding CEA and luciferase to obtain the A-431CEA-luc cell line, as previously described [14 (link)]. HER1- and HER2-expressing SK-OV-3 cells were transfected with the gene encoding CEA to obtain the SK-OV-31B9 cell line. All cell lines were grown in DMEM supplemented with 10% heat-inactivated foetal bovine serum, 1% penicillin/streptomycin, and 200 µg/mL geneticin. Hygromycin (100 µg/mL) was added to the medium for A-431CEA-luc cells. Cell lines were routinely tested for mycoplasma contamination using the MycoTect assay from Life technologies (Thermo Fisher Scientific, Waltham, MA, USA).
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