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8 protocols using skbr3 htb 30

1

Culturing Diverse Cancer Cell Lines

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Raji (CCL-86), ARH-77 (CRL-1621), Daudi (CCL-213) and SK-BR-3 (HTB-30) cells were purchased from ATCC (Manassas, VA, USA). Raji, ARH-77 and Daudi were propagated in RPMI with 10% fetal bovine serum (FCS) (both Sigma-Aldrich, St. Louis, MO, USA) with 4 mM glutamine, sodium pyruvate and penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in humidified atmosphere with 5% CO2 and diluted three times a week to be kept below 1 × 106 cells/mL. ARH-77 cells were detached with a cell-scraper (TPP, Trasadingen, Switzerland). SK-BR-3 cells were kept in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) with 10% FBS with 4 mM glutamine and penicillin/streptomycin and passaged twice a week by Trypsinization: the cells in a T75 flask were rinsed twice with 12 mL pre-warmed PBS, detached with 3 mL 0.1% Trypsin/0.05 M EDTA (Sigma-Aldrich, St. Louis, MO, USA) for 5 min at 37 °C, at 5% CO2 in humidified atmosphere, and collected in 9 mL DMEM with all additives with a centrifugation at 300× g, 5 min at RT before resuspension in fresh complete medium.
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2

Culturing Primary and Immortalized Breast Cells

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Primary human breast epithelial HMEC cells (CC-2551, Lonza, Basel, Switzerland) and immortalized breast epithelial 184A1 cells (a kind gift from A/Prof. Darren Shafren, the University of Newcastle) were cultured in mammary epithelial basal medium (MEBM) supplemented with bovine pituitary extract (BPE) (0.4%), human epidermal growth factor (hEGF) (0.1%), hydrocortisone (0.1%), GA-1000 (0.1%), and insulin (0.1%) (Lonza). MCF7 (HTB-22, American Type Culture Collection (ATCC), Manassas VA, USA), T-47D (HTB-133, ATCC), MDA-MB-231 (HTB-26, ATCC), and SKBR3 (HTB-30, ATCC; a kind gift from A/Prof. Darren Shafren, the University of Newcastle) breast cancer cell lines were cultured in RPMI-1640 (GE Healthcare, Chicago, IL, USA) supplemented with 10% FBS (Sigma–Aldrich, St. Louis, MO, USA) and 2 mM L-glutamine (GE Healthcare). All cells were maintained at 37 °C with 5% CO2 and used within four years of purchase from ATCC or Lonza, or authenticated using the GenePrint 10 System (Promega, Madison, WI, USA) as per the manufacturer’s instructions, and DNA fragments were detected by the Australian Genome Research Facility (AGRF) (Melbourne, VIC, Australia).
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3

Characterization of Breast Cancer Cell Lines

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The human breast cancer cell lines SKBR3 (HTB-30) and BT474 (HTB-20) were obtained from American Type Culture Collection. Monolayer cultures of the cells were maintained in DMEM/F12 medium with 10% fetal bovine serum. The cell lines overexpressed the HER2/c-erb-2 (HER2) gene product. The JIMT-1 cell line that was established from a breast cancer patient who clinically demonstrated resistance to trastuzumab was purchased from German Collection of Microorganisms and Cell Cultures. The JIMT-1 cell line also showed HER2 overexpression [20 (link)]. Both SKBR3 and JIMT-1 lacked expressions of estrogen and progesterone receptors (ER/PR) and BT474 expresses ER in addition to HER2 receptor.
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4

Breast Cancer Cell Line Culture

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Breast cancer cell lines (SK-BR-3 (HTB-30) and MCF7 (HTB-22)) and culture media were obtained from American Type Culture Collection (ATCC, USA). SK-BR-3 cells were cultivated in McCoy’s 5a Medium Modified (30–2007) supplemented with 10% fetal bovine serum (FBS, 164,210–500, Procell). MCF7 cells were cultured in Eagle’s Minimum Essential Medium (30–2003) added with 0.01 mg/ml human recombinant insulin (91077C, Sigma-Aldrich, USA) and 10% FBS. The incubation was carried out at 37°C with 5% CO2 (Heracell Vios 160i CR CO2 incubator, 51,033,770, Thermo Scientific, USA).
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5

Cell Line Culture and Maintenance

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All cell lines were cultured at 37°C under 5% CO2 in the appropriate medium containing 10% fetal bovine serum and penicillin-streptomycin (15070063, Thermo Fisher Scientific). Cell lines J.RT3-T3.5 (TIB-153), HEK293T (CRL-3216), HCT116 (CCL-247), C32TG(CRL-1579), HepG2 (HB-8065), A375 (CRL-1619), SK-Br-3 (HTB-30), Cama-1 (HTB-21), HeLa (CCL-2), Capan-1 (HTB-79), Capan-2, (HTB-80) and K562 (CCL-243) were obtained from the American Type Culture Collection. DLD-1 (90102540) was obtained from the European Collection of Authenticated Cell Cultures. SNG-M (JCRB0179) was obtained from the Japanese Collection of Research Bioresources Cell Bank. The Jurkat TCR/CD3 effector cell line (J129A) was obtained from Promega. All cell lines were maintained in an appropriate medium under conditions recommended by the supplier. Patient-derived xenograft (PDX) cells were obtained from CrownBio. PDX cells were thawed as per manufactures guidelines and seeded directly into assay plates.
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6

Breast Cancer Cell Line Maintenance

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The human breast cancer cell lines MCF-7 (HTB-22™), T47D (HTB-133™), MDA-MB-231 (HTB-26™) and SKBR3 (HTB-30™) were obtained from the American Type Culture Collection (ATCC, LGC Standards GmbH, Wesel, Germany) and maintained in Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (DMEM/F12 without phenol red, Thermo Fisher Scientific, Rockford, IL, USA), supplemented with 10% FCS (Thermo Fisher Scientific, Rockford, IL, USA), 50 units/mL penicillin and 50 µg/mL streptomycin sulfate (Invitrogen AG, Carlsbad, CA, USA) at 37 °C with 5% CO2. All cell lines were authenticated (Multiplexion, Heidelberg, Germany) and negatively tested for mycoplasma contamination before and after completion of the study.
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7

Characterization of HER2+ Breast Cancer Cell Lines

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HER2+ breast cancer cell lines BT474 (HTB-20) representing Luminal B (ER+; PR+, HER2+; ductal carcinoma) subtype and SKBR3 (HTB-30) representing HER2+ subtype (ER-; PR-; HER2+; adenocarcinoma) of breast cancer were purchased from the American Type Culture Collection (ATCC) [25 (link),26 (link)], which were known to well characterized in previous studies and decribed highly sensitive to trastuzumab treatment [27 (link),28 ]. SKBR3 cells were maintained in Mc Coy’s 5A medium (Lonza) with L-glutamine containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin. BT474 cells were maintained in RPMI 1640 medium with L-glutamine (Lonza) supplemented with 10% FBS, 1% penicillin-streptomycin and 2% bovine insulin. The cell lines were cultured in a humidified air supplemented with 5% CO2 at 37°C. Trastuzumab was (kindly gifted by Prof. Dr. Hakan Gürdal from the Department of Pharmacology in Medical School of Ankara University) dissolved in phosphate-buffered saline (PBS) (stock concentration of 300 mg/mL) and stored at 4°C.
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8

Evaluating Cell Proliferation and Invasion

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HCC1954 (CRL-2338™) and SKBR3 (HTB-30™) cells were purchased from the American Type Culture Collection (ATCC®). siRNA transfections were performed using Lipofectamine® RNAiMAX (Invitrogen) according to the manufacturer’s guidelines. Proliferation kinetics and tumorspheres were recorded using Celigo® S imaging cytometer (Nexcelom Bioscience LLC) and IncuCyte® Live Cell Analysis System (Sartorius AG). Colonies and migrated cells from transwell assay were washed with PBS, fixed, stained and scanned with an Epson Perfection V700 Photo. Further details are available in Supplementary Data.
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