Murine embryonic fibroblast 3T3-L1 cells (ATCC, #CL-173™) were maintained in a proliferative medium (PM) made up of DMEM high glucose (4.5 g/L glucose), supplemented with 10% newborn calf serum, 4 mM L-glutamine, 1 mM, sodium pyruvate and 1% antibiotic–antimycotic cocktail (100 U/mL penicillin, 10 µg/mL streptomycin, 0.25 µg/mL amphotericin B). Murine PCa cells TRAMP-C1 (ATCC, #CRL-2730™) were cultured in DMEM high glucose supplemented with 5% fetal bovine serum (FBS), 4 mM L-glutamine, 5% Nu-serum IV complement, 0.005 mg/mL insulin, 10 nM dehydroepiandrosterone (DHEA) and 1% of an antibiotic–antimycotic cocktail. Human androgen-dependent LNCaP cells (ATCC, #CRL-1740) were cultured in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 15 mM HEPES, and 1% of antibiotic cocktail (100 U/mL penicillin, 10 µg/mL streptomycin). Human androgen-independent PC-3 cells (ATCC, #CRL-1435) were cultured in DMEM/F12 medium supplemented with 10% FBS, 2 mM L-glutamine, and 1% of an antibiotic–antimycotic cocktail. All cell lines were cultured at standard conditions (Tª: 37 oC, CO2: 5%). FBS was stripped from small weight molecules, including steroids, by using a protocol previously published [16 (link)]. Charcoal-stripped FBS (csFBS) was employed to perform a cell culture medium without androgens as a substitute of FBS.
Crl 1740
Manufactured by American Type Culture Collection
Sourced in United States
CRL-1740 is a cell line derived from human embryonic kidney cells. It is commonly used for a variety of cell culture applications in research and development.
Automatically generated - may contain errors
Lab products found in correlation
Lncap, by American Type Culture Collection (13 mentions)
Crl 1435, by American Type Culture Collection (8 mentions)
Rpmi 1640, by Thermo Fisher Scientific (5 mentions)
Crl 2505, by American Type Culture Collection (4 mentions)
Crl 11609, by American Type Culture Collection (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Lncap cell, by American Type Culture Collection (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Crl 3216, by American Type Culture Collection (3 mentions)
Htb 81, by American Type Culture Collection (3 mentions)
Du145, by American Type Culture Collection (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Pcs 440 010, by American Type Culture Collection (2 mentions)
Nlp005005mg, by PerkinElmer (2 mentions)
Keratinocyte serum free medium, by Thermo Fisher Scientific (2 mentions)
Lt07 318, by Lonza (2 mentions)
22rv1 cell, by American Type Culture Collection (2 mentions)
38 protocols using crl 1740
1
Culturing Murine and Human Prostate Cancer Cell Lines
2
Culturing Multiple Cell Lines
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Normal human bronchial epithelial cells (ATCC® PCS-300-010™, VA, USA), prostate epithelial cells (ATCC PCS-440-010™), mammary epithelial cells (ATCC PCS-600-010™), A549 (human lung tumor cells, ATCC CCL-185™), LNCaP (human prostate adenocarcinoma cells, ATCC CRL-1740™) and T-47D (human breast tumor cells, ATCC HTB133™) were used. Cells were cultured in appropriate cell growth kit, supplemented with additional growth factors as provided by ATCC, and maintained in a 5% CO2 humidified incubator at 37°C.
3
Culturing Human Prostate Cancer Cell Lines
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Human PC cell lines (PC346, LNCap, MDAPC1 2a/b, C4-2, PC3, and DU145) were obtained from the American Type Culture Collection (Catalog number CRL-2876, CRL-1740, CRL-2242, CRL-3314, CRL-1435, and HTB-81, respectively, ATCC, Rockville, MD, USA). Human PC cell line (BPH1) was obtained from Accegen Biotechnology (Catalog number ABC-TC454S, Fairfield, NJ, USA). All lines were maintained in DMEM (Invitrogen, Rockville, MD, USA) suppled with 5% fetal bovine serum (FBS; Sigma-Aldrich, Rockville, MD, USA) in a humidified incubator at 37 °C with a 5% CO2 atmosphere.
4
Prostate Cancer Cell Authentication
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LNCaP human prostate cancer cells (CRL-1740™, ATCC) were cultured in standard cell culture conditions (+37 °C, 5% CO2) until semiconfluent and authenticated using short tandem repeat analysis (GenePrint10 system, Promega, Madison, WI, USA) at the Institute for Molecular Medicine Finland (FIMM, Helsinki, Finland). The cells were tested negative for mycoplasma contamination in-house and for murine pathogens by IDEXX Laboratories Inc. (Ludwigsburg, Germany).
5
Prostate Cancer Cell Line Cultivation
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Human prostate PC-3 (CRL-1435, American Type Culture Collection (ATCC), Rockville, MD, USA), LNCaP (ATCC, CRL-1740) and DU 145 (ATCC, HTB-81) cells were cultured as monolayers in 50% Dulbecco’s modified Eagle’s medium (DMEM) and 50% Ham’s F12, RPMI1640 and DMEM, respectively, supplemented with 10% fetal calf serum. PZ-HPV-7 cells (ATCC, CRL-2221), an immortalized cell line derived from normal human prostate cells, were cultured in keratinocyte-serum free medium supplemented with 5 ng/ml human recombinant epidermal growth factor and 0.05 mg/ml bovine pituitary extract. The benign prostatic Hyperplasia BPH-1 cells were kindly provided by Prof. J. Swinnen (KU Leuven, Belgium) and were maintained in RPMI medium 1640 plus 10% fetal calf serum [15 (link)].
6
Androgen Regulation of LNCaP Cells
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Cell culture and androgen treatment of cells was as described previously [10 (link)-12 (link)]. All cells were grown at 37°C in 5% CO2. LNCaP cells (CRL-1740, ATCC) were maintained in RPMI-1640 with L-Glutamine (PAA Laboratories, R15-802) supplemented with 10% Fetal Bovine Serum (FBS) (PAA Laboratories, A15-101). For androgen treatment of LNCaP cells, medium was supplemented with 10% dextran charcoal stripped FBS (PAA Laboratories, A15-119) to produce a steroid-deplete medium. Following culture for 72 hours, 10nM synthetic androgen analogue methyltrienolone (R1881) (Perkin–Elmer, NLP005005MG) was added (Androgen +) or absent (Steroid deplete) for the times indicated. Where indicated, LNCaP cells were pre-treated for 1 hour with vehicle (dimethylsulfoxide; DMSO) (Sigma, C1988) or 1 μg/ml cycloheximide (Sigma, D2438) prior to addition of 10 nM R1881 for 24 hours as previously described [16 (link)]. Flp-In™-293 cells (R750-07, Invitrogen) were maintained in DMEM GlutaMax (Invitrogen, 10566-040), supplemented with 10% FBS (PAA Laboratories, A15-101) and stable cell lines generated using the Flp-In T-Rex Core Kit (K6500-01, Invitrogen) according to the manufacturer's instructions. Protein expression was induced using 1 μg/ml tetracycline (T7660, Sigma) for 72 hours.
7
Culturing Prostate Cancer and Immune Cells
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The prostate cancer cell lines LNCaP (ATCC CRL-1740) and LNCaP-abl (ATCC CVCL-4793) the monocytic cell line THP-1 (ATCC TIB-202) and immortalized foreskin fibroblast BJ cell line (CRL-2522) were cultured in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin-streptomycin (P/S, Gibco). For hormonal related experiments all cells were cultured in RPMI 1640 supplemented with 5% Dextran Coated Charcoal (DCC, Sigma) stripped-serum and 1% P/S 3 days before to the start of the experiment. AR was induced with 10 nM R1881 (Sigma) supplemented RPMI-DCC. Cell lines were kept at low passage and regularly tested mycoplasma negative. THP-1 cells were stimulated with either 100 ng/mL (for M1 macrophages) or 50 ng/mL (for M2 macrophages) of phorbol 12-myristate 13-acetate (PMA, Sigma) for 48 h, followed by 24 h in fresh 10%FBS-RPMI. M1-macrophages were differentiated by 24 h stimulation of 10 ng/mL lipopolysaccharide (LPS, Sigma) and 10 ng/mL interferon-γ (IFN-γ, Peprotech), while M2-macrophages were differentiated by 72 h stimulation with 20 ng/mL IL-4 (Peprotech) and 20 ng/mL IL-13 (Peprotech).
8
Stable Knockdown of PDE4D7 in LNCaP Cells
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LNCaP clone FGC (ATCC® CRL1740™) cells were cultured in RPMI 1640 medium (Gibco™) supplemented with 10% (v/v) foetal bovine serum, 1% (v/v) l -glutamine and 1% (v/v) penicillin/streptomycin. The lentiviral transfer plasmid was designed to stably express an shRNA hairpin targeting the first coding exon of the PDE4D7 transcript. As a control, a scrambled nucleotide sequence of the PDE4D7-targeting shRNA was cloned into the transfer plasmid. LNCaP FGC cells were infected with lentiviruses at MOI = 10 in the presence of 10 µg/ml polybrene. Cells were maintained in a puromycin-supplemented medium prior to selection and clonal expansion of stably transduced cells (Supplementary Materials ).
9
LNCaP Cell Culture Protocol
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LNCaP cells were purchased from ATCC® CRL-1740™ (Manassas, VA, USA) and grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco™, Thermo Fischer Scientific, Waltham, MA, USA) and supplemented with 10% fetal calf serum (FCS) (Gibco™, Thermo Fischer Scientific, Waltham, MA, USA) and 5% penicillin-streptomycin (Gibco™, Thermo Fischer Scientific, Waltham, MA, USA). Cells were cultured in 150 cm3 tissue culture flasks until they were about 75–80% confluent. Then, the cells were washed with phosphate-buffered saline (PBS) (Gibco™, Thermo Fischer Scientific, Waltham, MA, USA), trypsinized and resuspended in 1 mL RPMI medium without supplements. Cells were grown at 37 °C and 5% CO2.
A 0.4% trypan blue solution (Gibco™, Thermo Fischer Scientific, Waltham, MA, USA) was used to count the cells.
A 0.4% trypan blue solution (Gibco™, Thermo Fischer Scientific, Waltham, MA, USA) was used to count the cells.
10
Cell Line Culture Conditions
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The human-derived metastatic prostate carcinoma cell line LNCaP was obtained from American Type Culture Collection (CRL-1740, ATCC®, Manassas, VA, USA). The human prostate cancer cell line PC3 was provided by the Department of Biomedical and Biotechnological Sciences, University of Catania, Italy. LNCaP cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium with a GlutaMAX™ supplement and supplemented with fetal bovine serum 10% (v/v) (FBS) and antibiotics (100 units/mL penicillin, and 100 μg/mL streptomycin).
Routine maintenance of PC3 cells was in Dulbecco’s Modified Eagle’s Medium (DMEM) medium supplemented with 10% (v/v) FBS and antibiotics (100 units/mL penicillin, and 100 μg/mL streptomycin).
Human foreskin fibroblasts (HFF-1), obtained from the American Type Culture Collection (SCRC-1041, ATCC®, Rockville, MD, USA), were cultured in (DMEM) supplemented with 15% (FBS), 4.5 g/L glucose, 100 units/mL penicillin, and 100 μg/mL streptomycin. All the cells were cultured in a 5% CO2-equilibrated 37 °C incubator.
Routine maintenance of PC3 cells was in Dulbecco’s Modified Eagle’s Medium (DMEM) medium supplemented with 10% (v/v) FBS and antibiotics (100 units/mL penicillin, and 100 μg/mL streptomycin).
Human foreskin fibroblasts (HFF-1), obtained from the American Type Culture Collection (SCRC-1041, ATCC®, Rockville, MD, USA), were cultured in (DMEM) supplemented with 15% (FBS), 4.5 g/L glucose, 100 units/mL penicillin, and 100 μg/mL streptomycin. All the cells were cultured in a 5% CO2-equilibrated 37 °C incubator.
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