MDA-MB-231 breast cancer cells38 (link) (HTB-26, ATCC, Manassas, VA), MCF-7 breast cancer cells39 (link) (HTB-22, ATCC), ZR75-1 breast cancer cells40 (link) (CRL-1500, ATCC), PC-3 prostate cancer cells41 (link) (CRL-1435, ATCC), and RWGT2 lung cancer cells15 (link),42 (link) (isolated from bone metastases by Dr. Guise as reported15 (link)) were cultured in Dulbecco's modified Eagle's media (DMEM) (Hyclone, Logan, UT) containing 10% heat-inactivated fetal bovine serum (FBS) (Hyclone). JJN-3 multiple myeloma cells43 (link) (ACC 541, DSMZ) were cultured in RPMI 1640 (Invitrogen, Grand Island, NY) containing 10% heat-inactivated FBS. A549 cancer cells (CCL-185, ATCC) were cultured in 1640 RPMI (Hyclone) containing 10% heat-inactivated FBS. C2C12 myoblast cells (CRL-1772, ATCC) were cultured subconfluently in DMEM containing 10% heat-inactivated FBS. C2C12 myoblasts were differentiated into myotubes by culture in DMEM containing 2% heat-inactivated horse serum (HS) (Hyclone). All cells were maintained at 37° C with 5% CO2 in a humidified chamber. All cells were verified to be free of mycoplasma contamination via routine PCR testing. No independent verification was completed. Cells treated with recombinant human TGF-β1 (R&D Systems, Minneapolis, MN) were starved in DMEM (no serum) for 16-20 hrs and 5 ng/ml TGF-β1 was added to cells in DMEM.
Crl 1500
Manufactured by American Type Culture Collection
Sourced in United States
The CRL-1500 is a laboratory equipment product offered by the American Type Culture Collection (ATCC). It is designed to facilitate the growth and maintenance of cell cultures. The core function of the CRL-1500 is to provide a controlled environment for cell cultivation, supporting the in vitro propagation of various cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Htb 26, by American Type Culture Collection (4 mentions)
Htb 22, by American Type Culture Collection (3 mentions)
Recombinant human tgf β1, by R&D Systems (2 mentions)
Crl 1435, by American Type Culture Collection (2 mentions)
Crl 1772, by American Type Culture Collection (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (2 mentions)
Tgf β1, by R&D Systems (2 mentions)
Human insulin, by Thermo Fisher Scientific (1 mentions)
Zr 75 1, by American Type Culture Collection (1 mentions)
Rpmi 1640 medium, by American Type Culture Collection (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Ultra low attachment plate, by Corning (1 mentions)
Gentamycin, by Thermo Fisher Scientific (1 mentions)
Htb 22d, by American Type Culture Collection (1 mentions)
Htb 129, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Shc 202v, by Merck Group (1 mentions)
Crispr12v, by Merck Group (1 mentions)
6 protocols using crl 1500
1
Culturing and Differentiating Cancer Cell Lines
2
Culturing and Differentiating Cancer Cell Lines
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MDA-MB-231 breast cancer cells38 (link) (HTB-26, ATCC, Manassas, VA), MCF-7 breast cancer cells39 (link) (HTB-22, ATCC), ZR75-1 breast cancer cells40 (link) (CRL-1500, ATCC), PC-3 prostate cancer cells41 (link) (CRL-1435, ATCC), and RWGT2 lung cancer cells15 (link),42 (link) (isolated from bone metastases by Dr. Guise as reported15 (link)) were cultured in Dulbecco's modified Eagle's media (DMEM) (Hyclone, Logan, UT) containing 10% heat-inactivated fetal bovine serum (FBS) (Hyclone). JJN-3 multiple myeloma cells43 (link) (ACC 541, DSMZ) were cultured in RPMI 1640 (Invitrogen, Grand Island, NY) containing 10% heat-inactivated FBS. A549 cancer cells (CCL-185, ATCC) were cultured in 1640 RPMI (Hyclone) containing 10% heat-inactivated FBS. C2C12 myoblast cells (CRL-1772, ATCC) were cultured subconfluently in DMEM containing 10% heat-inactivated FBS. C2C12 myoblasts were differentiated into myotubes by culture in DMEM containing 2% heat-inactivated horse serum (HS) (Hyclone). All cells were maintained at 37° C with 5% CO2 in a humidified chamber. All cells were verified to be free of mycoplasma contamination via routine PCR testing. No independent verification was completed. Cells treated with recombinant human TGF-β1 (R&D Systems, Minneapolis, MN) were starved in DMEM (no serum) for 16-20 hrs and 5 ng/ml TGF-β1 was added to cells in DMEM.
3
Culturing Human Breast Cancer Cell Lines
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Human breast cancer cell lines of MCF7 (ATCC, HTB-22), ZR75-1 (ATCC, CRL-1500), and MDA-MB-231 (ATCC, HTB-26) were obtained from the American Type Culture Collection (Manassas, VA, USA) and sub-cultured, as per supplier’s instructions. MCF7 cells were cultured in Eagle’s Minimum Essential Medium (EMEM) (ATCC) supplemented with 10% fetal bovine serum (FBS) (Corning, Corning, NY, USA), and Human Insulin (Invitrogen, Carlsbad, CA, USA). ZR75-1 cells were cultured in the RPMI-1640 Medium (ATCC, 30-2001), with 10% FBS, and the MDA-MB-231 cells were cultured in the DMEM/F-12 (1:1) medium (Mediatech, Herndon, VA, USA), containing 5% FBS, 4 mM glutamine, 50 µM β-Mercaptoethanol, and 1 mM sodium pyruvate. Cell lines were maintained at 37 °C with 5% CO2. For the suspension culture, cells were seeded into Corning Ultra-Low Attachment plates, which inhibits immobilization to the surface. Cells were maintained in suspension with media changes every other day, for up to seven days.
4
Characterization of Breast Cancer Cell Lines
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ZR-75-1: This cell line composed of adherent epithelial estrogen dependent breast cancer cells (ATCC, CRL-1500). They have a more circular structure than other breast cancer cell lines and tend to grow as monolayer with mosaic shaped clusters in culture.
MCF-7: These are epithelial cells derived from a human breast carcinoma (ATCC, HTB-22D). They tend to grow into clusters which, leads to mosaic.
CAMA-1: Isolated as adherent patches of epithelial cells from malignant pleural effusion derived from a human breast carcinoma (ATCC, HTB-21). It grows as compost, multilayered colonies in culture.
MDA-MB-435: This cell line was derived from pleural effusion and cells appear morphologically epithelial like and are considered to be highly metastatic (ATCC, HTB-129). All the breast carcinoma cell lines have been grown in Rosewell Park Memorial Institute 1640 (RPMI) (Invirogen, USA) with ultra-glutamine 1: Supplemented with 10% (v/v) heat activated FBS (Sigma, USA) and 0.5% (v/v) antibiotic gentamycin (Gibco, BRL). Cell lines were maintained at 37°C in an atmosphere of 5% CO2.
5
Establishing Bone Metastasis Model
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With approval from the University Health Network animal care committee (AUP 6044), human tumor cells were injected into 5-week old athymic female Hsd:RH-Foxn1rnu rats (Envigo, Indianapolis, IN, USA) through intracardiac injection under general inhalation anaesthesia. The intracardiac injection of luciferase transfected human ZR-75-1 (ATCC® CRL-1500™) breast cancer cells resulted in the development of osteoblastic metastases within 56 days. Intra-cardiac injection of 1.5 x 106 luciferase transfected HeLa cells [33 (link), 34 (link)] produced osteolytic bone metastases within 14 days in the athymic rats. All the rats injected in this study developed metastases at the presented timelines (Fig 1 ). The different age of the rats at onset of bone metastases (osteoblastic vs osteolytic) required the use of age-matched healthy animals due to age-dependent differences in bone remodeling.
6
MOSPD2 Silencing in Breast Cancer Cells
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Human breast cancer cell lines MDA‐MB‐231 (hereafter MDA‐231) (ATCC® HTB‐26™), BT‐20 (ATCC® HTB19™) and ZR‐75‐1 (ATCC® CRL‐1500™) were purchased from ATCC. The cells were used in the described experiments between 2 and 6 weeks from thawing. Cells (2 × 106 in 2 mL) were placed in a 15 mL tube and lentiviral particle‐expressing control short hairpin RNA (sh‐Control) (SHC202V, Sigma, Israel) or human MOSPD2 (sh‐MOSPD2) Exon 4 (TRCN0000323142 Sigma) were applied on the cells, which were then spun for 60 min at 2,000 rpm at room temperature in the presence of 8 μg/mL polybrene (Sigma). The cells were then seeded in a 6‐well plate. After 72 hr, fresh medium containing puromycin (4 μg/mL Sigma) was added for the selection of transduced cells. For CRISPR‐CAS9 (hereafter CRISPR)‐mediated silencing, cells were transduced with CRISPR non‐target control (CRISPR‐Control) (CRISPR12V, Sigma), CRISPR human MOSPD2 Exon 3 (CRISPR‐MOSPD2) (HS0000528665, Sigma) or Exon 9 (HS0000176080, Sigma) lentiviral particles as described above. Single‐cell cloning was performed on CRISPR‐transduced cells to isolate cells with silenced MOSPD2 protein expression.
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