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8 protocols using lncap human prostate cancer cell line

1

Prostate Cancer Cell Line Cultivation and Authentication

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The androgen-sensitive LNCaP human prostate cancer cell line was purchased from ATCC (Chicago, IL, USA). The LNCaP cells were maintained in an RPMI 1640 medium, supplemented with 10% (v:v) fetal bovine serum, 100 IU/mL of penicillin, and 100 µg/mL of streptomycin at 37 °C in a 5% CO2 incubator. The WPE1-NA22 cell line was derived from a non-tumorigenic human prostate epithelial cell line, RWPE-1, after exposure to a chemical carcinogen, N-methyl-N-nitrosourea, and selected and cloned in vivo and in vitro [30 (link)]. It mimics a pre-malignant stage of prostatic intra-epithelial neoplasia (PIN). The WPE1-NA22 cells were purchased from ATCC and cultured in a keratinocyte serum-free medium supplemented with 0.05 mg/mL of bovine pituitary extract and 5 ng/mL of epidermal growth factor (Invitrogen, Carlsbad, CA, USA). Normal prostate epithelial PrEC cells were purchased from Lonza Walkersville, Inc. (Walkersville, MD, USA) and cultured in a PrEGM medium (Lonza Walkersville, Inc.). All cell lines were tested periodically for mycoplasma contamination using a PCR-based Universal Mycoplasma Detection kit (ATCC).
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2

Culturing Androgen-Dependent Prostate Cancer Cells

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The androgen-dependent LNCaP human prostate cancer cell line was purchased from ATCC (Chicago, IL, USA). Androgen-dependent LAPC-4 cell line is a gift from Dr. Charles Sawyers' laboratory previously at UCLA. Both cell lines were maintained in RPMI 1640 medium, supplemented with 10% (v:v) of fetal bovine serum (FBS), 100 IU/mL of penicillin and 100 μg/mL of streptomycin at 37 °C in a 5% CO2 incubator.
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3

Prostate Cancer Cell Line Assay

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LNCaP human prostate cancer cell line was obtained from ATCC (Manassas, VA, USA). Salmonella typhimurium strain TA1535/pSK1002 was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Inhoffenstraße 7B 38124 Braunschweig, Germany). DMEM medium supplemented with phenol red, Ultraglutamine and high glucose (4.5 g/L) was from Lonza (Verviers, Belgium). The solutions of penicillin/streptomycin, 0.25% trypsin-EDTA and phosphate buffered saline (PBS) were from Gibco (Paisley, UK). Genistein (≥98%, HPLC), daidzein (≥98%), equol (≥99%, TLC), fetal bovine serum (FBS), sodium pyruvate solution (100 mM), deoxyribonucleic acid sodium salt type XIV: from herring testes, bisbenzimide H 33258, camptothecin, 4-nitroquinoline N-oxide (4NQO), 2-aminoanthracene (2AA), d-glucose 6-phosphate disodium salt hydrate (G-6-P) and 2-nitrophenyl β-d-galactopyranoside (ONPG) were purchased from Sigma-Aldrich (Poznań, Poland). Nicotinamide adenine dinucleotide phosphate (NADP) was purchased from MP Biomedicals. Zinc sulphate (ZnSO4 × 7H2O, ≥99.5%), sodium selenate (Na2SeO4, ≥98%), copper(II)/cupric sulphate (CuSO4 × 5H2O, ≥99%), iron(II) sulphate (FeSO4 × 7H2O, ≥99%), DMSO were obtained from POCH (Gliwice, Poland) and calcium chloride (CaCl2 × 6H2O, ≥98%) was from Chempur (Piekary Śląskie, Poland). Methanol was purchased from Merck (Warsaw, Poland).
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4

Prostate Cancer Cell Lines and Transfection

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LNCaP human prostate cancer cell line was obtained from the American Type Culture Collection. Early-passage cells were used in all experiments. These cells were grown in T-medium (Invitrogen, Grand Island NY, USA) supplemented with 10% FBS, 100 units/ml penicillin, and 100 ug/ml streptomycin at 37°C with 5% CO2. M12 cells are AR-negative and were obtained from Dr. J. Ware at the Medical College of Virginia. The generation and characterization of the M12 prostate cell line has been described previously [50 (link)–53 (link)]. M12 cells were cultured in RPMI 1640 medium (Invitrogen) containing 5% FBS, 10 ng/ml EGF, 0.02 mM dexamethasone, 5 ug/ml insulin, 5 ug/ml transfection, 5 ng/ml selenium, fungizone, and gentamicin at 37°C with 5% CO2. cDNA of the entire ARv567es variant and ARfl were cloned into p3xFlag-CMV-9 vector as described previously [19 (link), 53 (link)]. The expression constructs were transfected into the human prostate cancer cell lines using TurboFect reagent according to the manufacturer's protocol (Thermo Scientific, Pittsburgh PA, USA).
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5

Culturing LNCaP Prostate Cancer Cells

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The LNCaP human prostate cancer cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI 1640 medium with phenol red containing 10% fetal bovine serum (FBS) and 1% pen–strep at a concentration of 2.5 × 105 cells/mL in 175 cm2 flasks at 37 °C with 5% CO2.
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6

Apogossypol and Gossypol Inhibit LNCaP Prostate Cancer

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The LNCaP human prostate cancer cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI1640 medium (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL) and 1% penicillin/streptomycin in a humidified incubator at 37°C with 5% CO2. Apogossypol and gossypol were synthesized and extracted in our laboratory (25 (link)), dissolved in dimethyl sulfoxide (DMSO) and stored at ‑20°C. Working solutions were prepared by diluting the stock solution with culture medium before use. MTT was purchased from Sigma-Aldrich (St. Louis, MO, USA). The anti-Bcl-2, anti-caspase-3, and anti-caspase-8 antibodies were purchased from Maixin Biotechnology (Fuzhou, China), Zhongshan Golden Bridge Biotechnology (Beijing, China) and Boster Biological Engineering (Wuhan, China), respectively. Monkey anti-mouse immunoglobulin (Ig)G labeled with fluorescein isothiocyanate (FITC) and goat anti-rabbit IgG labeled with rhodamine were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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7

Prostate Cancer Cell Line Treatment

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LNCaP human prostate cancer cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI 1640 medium supplemented with a 10% heat-inactivated fetal bovine serum, HEPES (10 mM), penicillin, and streptomycin in a humidified 5% CO2 atmosphere at 37 °C. To examine the effects of TP, cells were cultured in phenol red-free RPMI 1640 containing 5% charcoal stripped serum (CSS) (TCB, Long Beach, CA, USA) for 24 h, and 100 nM TP was then added to the medium. STO-609 (a CaMKKβ inhibitor, Tocris Bioscience, Ellisville, MO, USA) was used at 30 μM and BAPTA-AM (a calcium chelator, Sigma-Aldrich, St. Louis, MO, USA) was used at 20 μM for the experiment.
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8

Prostate Cancer Cell Lines Response to IFNγ

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DU145 (both variants herein termed DU-L [E-cadherin low expressing] and DU-H [E-cadherin high expressing]), PC3 (both variants herein termed PC3-L [E-cadherin low expressing] and PC3-H [E-cadherin high expressing]) and LnCaP human prostate cancer cell lines, and RWPE-1 normal prostate cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). DU-H/DU-L and PC3-H/PC3-L cells were maintained in DMEM and F-12 K media, respectively. LnCaP cells were maintained in RPMI 1640 media and RWPE-1 cells in Keratinocyte Serum Free Medium (K-SFM). Cells were seeded into 6-well plates and after 24 h treated with IFNγ (5 ng/mL) or control (PBS) for 48 h. Cells were harvested for immunoblot or flow cytometry, or fixed for immunofluorescence.
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