Lncap

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The LNCaP is a cell line derived from a human prostate cancer metastatic site. It is a commonly used model for studying prostate cancer biology and testing potential therapeutic agents.

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274 protocols using lncap

Culturing and Overexpressing NRP1 in PCa Cells

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PCa cell lines (VCaP, LNCaP, 22Rv1, PC3 and DU145) were obtained from American Type Culture Collection and grown in RPMI-1640 (22Rv1, PC3, DU145, C4-2), DMEM (VCaP), or DMEM/RPMI (LNCaP) supplemented with 10% foetal bovine serum (FBS) (ThermoFischer Scientific, France). LNCaP-NE cells were obtained from LNCaP cells cultured in androgen-reduced conditions (phenol red-free DMEM/RPMI supplemented with 10% charcoal-stripped serum (CSS)).
Overexpression of NRP1 was obtained by stably transfecting LNCaP, C4-2 and 22Rv1 cell lines with the pCherry-mNrp1 plasmid (Addgene plasmid # 21934; [23 (link)] using lipofectamine 2000 (Invitrogen) standard protocol. Cells selection was performed in a medium containing G418 (300 µg/ml for C4-2 cells and 400 µg/ml for LNCaP and 22Rv1).

Blanc C., Moktefi A., Jolly A., de la Grange P., Gay D., Nicolaiew N., Semprez F., Maillé P., Soyeux P., Firlej V., Vacherot F., Destouches D., Amiche M., Terry S., de la Taille A., Londoño-Vallejo A., Allory Y., Delbé J, & Hamma-Kourbali Y. (2022). The Neuropilin-1/PKC axis promotes neuroendocrine differentiation and drug resistance of prostate cancer. British Journal of Cancer, 128(5), 918-927.

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Establishment of Docetaxel-Resistant PCa Cell Lines

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PCa cell lines, C4-2B and LNCaP, were purchased from American Tissue Culture Collection (ATCC, Manassas, VA, USA). C4-2B cells were maintained in DMEM plus F12 Medium (Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), insulin, triiodo-L-thyronine, transferrin, D-biotin, adenine (Millipore Sigma, St. Louis, MO, USA), and 10,000 U/mL penicillin/10,000 µg/mL streptomycin antibiotic solution (Fisher Scientific). LNCaP cells were maintained in RPMI-1640 media supplemented with 10% FBS, nonessential amino acids, HEPES, 2 mM L-glutamine, and penicillin/streptomycin antibiotic solution (Fisher Scientific). Docetaxel (DTX)-resistant cell lines were generated from parental C4-2B and LNCaP cells by gradually increasing the DTX concentration from 2 nM to 160 nM and were maintained in an incubator for 7 months at 37 °C and 5% CO₂. Likewise, resistant LNCaP cells were generated by gradually increasing the DTX concentration from 2 nM to 60 nM. The DTX-resistant cells, C4-2B-R and LNCaP-R, were maintained in RPMI-1640 media supplemented with 10% FBS, nonessential amino acids, HEPES, 2 mM L-glutamine, and penicillin/streptomycin antibiotic solution (Fisher Scientific). Both cell lines were maintained in an incubator at 37 °C and with 5% CO₂.

Singh S.K., Gordetsky J.B., Bae S., Acosta E.P., Lillard JW J.r, & Singh R. (2020). Selective Targeting of the Hedgehog Signaling Pathway by PBM Nanoparticles in Docetaxel-Resistant Prostate Cancer. Cells, 9(9), 1976.

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Prostate Cancer Cell Culture Conditions

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Cells were cultured at 37 °C in humidified air with 5% CO₂ under atmospheric oxygen pressure unless stated differently. LNCaP (ATCC® CRL-1740™, Manassas, VA) and PC-3 (ECCC, Wiltshire, UK), 22Rv1 (ATCC® CRL-2505™) and LNCaP-19 (castration-resistant LNCaP sub-line, characterized elsewhere)^{66 (link)} were cultured in RPMI-1640 (PAA Laboratories, Pasching, Austria) containing stable glutamine, supplemented with 1 mM sodium pyruvate and 10% fetal bovine serum (FBS; Gibco, South America) or 10% Dextran-Charcoal Stripped FBS (DCC; Gibco, South America), for LNCaP-19. The myofibroblast cell line WPMY-1 (ATCC® CRL-2854™) and VCaP (ATCC® CRL-2876™) was cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 5% and 10% FBS respectively. Sh-clones were cultured according to protocol for LNCaP. All cells were cultured with antibiotics (Penicillin-Streptomycin; Thermo Fisher Scientific) and routinely tested for mycoplasma contamination. In vitro experiments were conducted in biological triplicates if not stated differently.

Linder A., Hagberg Thulin M., Damber J.E, & Welén K. (2018). Analysis of regulator of G-protein signalling 2 (RGS2) expression and function during prostate cancer progression. Scientific Reports, 8, 17259.

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Prostate Cancer Cell Line Characterization and Transfection

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The human androgen‐dependent PCa cell line LNCaP (ATCC, Manassas, VA, USA) and three CRPC‐like cell lines including LNCaP‐AI, LNCaP‐Bic and C4‐2 (ATCC) were applied in this study. LNCaP and C4‐2 cells were maintained in RPMI‐1640 medium (Gibco, Waltham, MA, USA) containing 10% foetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco). LNCaP‐AI cells were grown in RPMI‐1640 without phenol red and supplemented with 10% charcoal‐stripped FBS (Gibco), whereas LNCaP‐Bic cells were added with 20 μM bicalutamide (Sigma, St. Louis, MO USA). Prior to any experiment, LNCaP cells were maintained in phenol red‐free medium containing charcoal‐stripped FBS for no less than 48 hours. All cells were incubated under a humidified atmosphere with 5% CO₂ at 37°C.
With respect to cell transfection, LNCaP‐AI and C4‐2 cells were transfected with indicated short hairpin RNAs (shRNAs), such as sh‐HNRNPA2B1#1/2, sh‐GOLGA8B#1/2, sh‐MAPK8IP3#1/2 and corresponding control sh‐NC, by use of Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) in line with manufacturers’ guide.

Cheng Y., Li L., Qin Z., Li X, & Qi F. (2020). Identification of castration‐resistant prostate cancer‐related hub genes using weighted gene co‐expression network analysis. Journal of Cellular and Molecular Medicine, 24(14), 8006-8017.

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Cell Culture Protocols for Prostate Cancer

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The cell lines used in this study included the human prostate cancer cells LNCaP (ATCC, Manassas, VA, USA), and the CRPC-like cell LNCaP-Bic and LNCaP-AI. LNCaP cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 (Gibco, Shanghai, China), supplemented with 10% FBS (fetal bovine serum, Shanghai ExCell Biology, China), LNCaP-AI cells were cultured in phenol red free RPMI-1640 containing 10% charcoal stripped FBS (Gibco, Shanghai, China); whereas LNCaP-Bic were cultured with 20 μM bicalutamide (Sigma, St. Louis, MI, USA). All media were supplemented with 1% penicillin/streptomycin. LNCaP cells were cultured in phenol red free medium with charcoal striped FBS for at least 2 days before any experiment. Cells were grown in a humidified atmosphere of 5% CO₂ at 37 °C. The construction of two CRPC LNCaP sublines was described in our previous article [10 (link)].

Gu P., Chen X., Xie R., Xie W., Huang L., Dong W., Han J., Liu X., Shen J., Huang J, & Lin T. (2019). A novel AR translational regulator lncRNA LBCS inhibits castration resistance of prostate cancer. Molecular Cancer, 18, 109.

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Culturing Diverse Prostate Cancer Cell Lines

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PrEC LH, LNCaP, C4-2, PC3, DU145, VCaP, NCI-H526, NCI-H660, and LASCPC-01 were purchased from ATCC (American Type Culture Collection). PrEC LH, LNCaP, C4-2, PC3, NCI-H526, VCaP and DU145 cells were cultured in RPMI 1640 (PrEC LH, LNCaP, C4-2, PC3, NCI-H526) or Dulbecco’s modified Eagle’s medium (VCaP, DU145) (Gibco, United States), supplemented with 10% fetal bovine serum (Invitrogen, United States) and 1% pen/strep (Gibco, United States). NCI-H660 and LASCPC-01 were cultured in RPMI 1640 medium, supplemented with 0.005 mg/ml Insulin, 0.01 mg/ml Transferrin, 30 nM Sodium selenite, 10 nM Hydrocortisone, 10 nM beta-estradiol, 4 mM l-glutamine (HyCloneTM, United States) and 10% FBS. Cells were maintained at 5% CO₂ in a 37 ℃ humidified incubator.

Wang Q., Chen J., Singh S., Xie Z., Qin F., Shi X., Cornelison R., Li H, & Huang H. (2022). Profile of chimeric RNAs and TMPRSS2-ERG e2e4 isoform in neuroendocrine prostate cancer. Cell & Bioscience, 12, 153.

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Cultivation of Prostate Cancer Cell Lines

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LNCaP (ATCC, Manassas, VA; CRL-1740), MDA-PCa-2b (ATCC, Manassas, VA; CRL-2422) and subline prostate cancer (PCa) cell lines were maintained in a 37°C, 5.0% (v/v) CO₂ growth chamber. LNCaP and LNCaP sublines, LNas1 and LNbs1, were cultured in DMEM (Gibco/Thermo Scientific; 1185–092) supplemented with 10% (v/v) fetal bovine serum essence (FBE; Seradigm; 3100–500), 0.4 mM L-glutamine (L-glut; Gibco/Invitrogen; 25030–081), and 10 U/ml penicillin G sodium and 10 mg/ml streptomycin sulfate (pen-strep; Gibco/Invitrogen; 15140–122). MDA-PCa-2b and MDA-PCa-2b sublines, MDA-αs1 and MDA-βs1, were cultured in BRFF-HPC1, (#0403, AthenaES, Baltimore, MD) supplemented with 20% (v/v) FB essence, 0.4mM L-glut, and 10 U/mL Gibco penicillin G sodium and 10 mg/mL streptomycin sulfate (pen-strep; #15140–122, Thermo Fisher Scientific).

Thomas-Jardin S.E., Kanchwala M.S., Dahl H., Liu V., Ahuja R., Soundharrajan R., Roos N., Diep S., Sandhu A., Xing C, & Delk N.A. (2021). Chronic IL-1 Exposed AR+ PCa Cell Lines Show Conserved Loss of IL-1 Sensitivity and Evolve Both Conserved and Unique Differential Gene Expression Profiles. Journal of cellular signaling, 2(4), 248-260.

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Culturing Drug-Resistant Prostate Cancer Cells

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The prostate cancer cell lines LNCaP and PC-3 were obtained from ATCC (American Type Culture Collection); LNCaP-abl was provided by Dr. Myles Brown’s laboratory. LAPC-4 cell line was originally acquired from University of California Los Angeles. LNCaP, PC-3, and LAPC-4 were maintained in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Sigma) and 1% penicillin-streptomycin. LNCaP-abl was cultured in phenol red-free RPMI 1640 medium supplemented with 5% charcoal striped FBS (cFBS) medium (Gibco) and 1% penicillin and streptomycin. All cells were grown at 37 °C in a humidified atmosphere at 5% CO₂. Drug-resistant prostate cancer cell lines were generated from parental LNCaP, LAPC-4, and LNCaP-abl cells by long-term culturing in the presence of increasing amounts of AA; established AA resistant cell lines, denoted LNCaP-AAr, LAPC4-AAr, and LNCaP-abl-AAr respectively, were routinely maintained in medium containing 5 μM AA. All cell lines are authenticated and routinely screened for contamination of mycoplasma.

Pan W., Zhang Z., Kimball H., Qu F., Berlind K., Stopsack K.H., Lee G.S., Choueiri T.K, & Kantoff P.W. (2021). Abiraterone acetate induces CREB1 phosphorylation and enhances the function of the CBP-p300 complex, leading to resistance in prostate cancer cells. Clinical cancer research : an official journal of the American Association for Cancer Research, 27(7), 2087-2099.

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Cultivation of Prostate Cancer Cell Lines

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Prostate Cancer Cell Culture Conditions

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Hormone na€ ve (HN) cells (CWR22res, LNCaP, and VCaP) were cultured in RPMI medium (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific) and 2 mmol/L glutamine (Gibco, Thermo Fisher Scientific). Castration-resistant cells (22rv1 and LNCaP AI) were cultured in androgen-deprived medium consisting of phenol-free RPMI (Gibco, Thermo Fisher Scientific) supplemented with 10% charcoal-stripped serum (CSS; Gibco, Thermo Fisher Scientific) and 2 mmol/L glutamine. All cells were kept in incubators set at 37 C and 5% CO 2 . LNCaP (ATCC CRL-1740), 22Rv1 (ATCC CRL-2505), and VCAP (ATCC CRL-2876) were obtained from ATCC. CWR22Res cells (hormone-responsive variant of CWR22 cells) were obtained from Case Western Reserve University, Cleveland, OH. LNCaP AI and CWR22Rv1-AR-EK cells were obtained from Newcastle University (Newcastle upon Tyne, United Kingdom). All cell lines were authenticated by short tandem repeat DNA profiling and were routinely tested for Mycoplasma (every $6 weeks) using the Mycoalert Mycoplasma detection kit (Lonza). Cells were kept in culture for a maximum of 10 passages after recovery from frozen vials.

Martinez R.S., Salji M.J., Rushworth L., Ntala C., Rodriguez Blanco G., Hedley A., Clark W., Peixoto P., Hervouet E., Renaude E., Kung S.H.Y., Galbraith L.C.A., Nixon C., Lilla S., MacKay G.M., Fazli L., Gaughan L., Sumpton D., Gleave M.E., Zanivan S., Blomme A, & Leung H.Y. (2021). SLFN5 Regulates LAT1-Mediated mTOR Activation in Castration-Resistant Prostate Cancer. Cancer research, 81(13).

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