The human PDAC cell lines BxPc-3, AsPC-1, PANC-1, and MIA-PaCa2 and the human hTERT-HPNE immortalized pancreatic duct cell line CRL-4023 (Cat#CRL-4023, RRID: CVCL_C466) were purchased from ATCC (Manassas, VA, USA), BxGEM cells are described [47 (link)], and the human hepatic stellate cell line LX2 was obtained from Merck (Darmstadt, Germany, Millipore Cat# SCC064, RRID:CVCL_5792). The cells were cultured as described [48 (link)].
Crl 4023
Manufactured by American Type Culture Collection
Sourced in United States
The CRL-4023 is a laboratory equipment item produced by American Type Culture Collection. It is designed for cell culture applications, but a detailed and unbiased description of its core function cannot be provided without potentially making interpretations or extrapolations.
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Lab products found in correlation
Aspc 1, by American Type Culture Collection (7 mentions)
Bxpc 3, by American Type Culture Collection (5 mentions)
Panc 1, by American Type Culture Collection (4 mentions)
Plasmotest, by InvivoGen (2 mentions)
Dmem high glucose, by Merck Group (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Mia paca 2, by American Type Culture Collection (2 mentions)
Crl 1687, by American Type Culture Collection (1 mentions)
Panc10, by American Type Culture Collection (1 mentions)
Pfu dna polymerase kit, by Promega (1 mentions)
Fetal bovine serum (fbs), by Sangon (1 mentions)
Dmem without glucose, by Merck Group (1 mentions)
Crl 10317, by American Type Culture Collection (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Medium m3 base, by INCELL (1 mentions)
Crl 1420, by American Type Culture Collection (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
10 protocols using crl 4023
1
Pancreatic and Hepatic Cell Lines
2
Cadmium Chloride Cytotoxicity in Pancreatic Cells
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The cell lines are obtained from the American Type Culture Collection (ATCC, Manassas, VA). Control pancreas cells [hTERT-HPNE (“human pancreatic Nestin-expressing” cells or HPNE; ATCC® CRL-4023™, control pancreatic cells)] and tumour [AsPC-1 (ATCC® CRL-1682™, pancreatic tumour cells), Panc-1 (ATCC #CRL-1469™, Pancreas ductal epithelioid carcinoma), Panc-10.05 (ATCC #CRL-2547™, Pancreatic epithelial adenocarcinoma), and BxPC-3 (ATCC #CRL-1687™, Pancreatic adenocarcinoma)] were grown and maintained as described in the ATCC-suggested protocols. Unless otherwise specified, cells were grown in their defined optimum growth media. For a parallel set of LC50 assays, cells were grown in a minimal media of MEM plus 1% foetal bovine serum (FBS). The cells were split in 6-well plates at 3–5 day intervals depending on confluence and cells were grown to 80% confluency in preparation for 14 days treatment with cadmium chloride (CdCl2; 50 μM), based on our previously published data (Djordjevic et al 2019 (link)).
3
Overexpression of Linc-pint in Pancreatic Cell Lines
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The human PDAC PL45 [cat. no. American Type Culture Collection (ATCC)® CRL-2558™] and normal human pancreatic ductal hTERT-HPNE (cat. no. ATCC® CRL-4023™) cell lines were purchased from ATCC (Manassas, VA, USA). Cells were cultured in ATCC-formulated Dulbecco's modified Eagle's medium (cat. no. 30-2002; ATCC) containing 10% fetal bovine serum (Sangon Biotech Co., Ltd., Shanghai, China) at 37°C in a humidified incubator containing 5% CO2.
PCR amplification was performed using Linc-pint primer with NheI cutting site at the 5′end to obtain full-length Linc-pint cDNA using Pfu DNA Polymerase kit (Promega Corporation, Madison, WI, USA). PCR product was subjected to agarose gel purification and was inserted into a linearized NheI-pEGFPC3 vector (Clontech Laboratories, Inc., Mountainview, CA, USA) to produce the Linc-pint expression vector. Cells from both cell lines were cultured for 8–12 h to reach 80–90% confluence. Cells (5×105) were then transfected with 10 nM vectors using Lipofectamine® 2000 reagent (cat. no. 11668-019; Invitrogen; Thermo Fisher Scientific, Inc.). Untransfected cells represented the control group. Cells transfected with empty vectors represented the negative control (NC) group. Experiments were performed 24 h following transfection.
PCR amplification was performed using Linc-pint primer with NheI cutting site at the 5′end to obtain full-length Linc-pint cDNA using Pfu DNA Polymerase kit (Promega Corporation, Madison, WI, USA). PCR product was subjected to agarose gel purification and was inserted into a linearized NheI-pEGFPC3 vector (Clontech Laboratories, Inc., Mountainview, CA, USA) to produce the Linc-pint expression vector. Cells from both cell lines were cultured for 8–12 h to reach 80–90% confluence. Cells (5×105) were then transfected with 10 nM vectors using Lipofectamine® 2000 reagent (cat. no. 11668-019; Invitrogen; Thermo Fisher Scientific, Inc.). Untransfected cells represented the control group. Cells transfected with empty vectors represented the negative control (NC) group. Experiments were performed 24 h following transfection.
4
Chronic Cadmium Exposure in Epithelial Cells
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We used two human non-cancer epithelial cell lines: one from breast (MCF10A), and one from pancreas (hTERT-HPNE). MCF10A were purchased from ATCC® (CRL-10317™) and were exposed with 2.5 µM Cd 2+ for 40 weeks according to previously published data (Benbrahim-Tallaa et al. 2009) (link). hTERT-HPNE were purchased by ATCC® (CRL-4023™) and exposed with 1 µM Cd 2+ for 30 weeks (Qu et al. 2012 (link)). MCF10A were cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% Fetal Calf Serum (FCS), hydroxycortisone (50 ng/mL), insulin (0.01 mg/mL), human recombinant EGF (20 ng/mL), and cholera toxin (100 ng/mL). hTERT-HPNE were cultured in base medium: 75% DMEM without glucose (Sigma) and 25% Medium M3 Base (Incell Corp) with 5% FCS. Complete growth medium was supplemented with the following components: human recombinant EGF (10 ng/mL), D-glucose (1 g/L), puromycin (750 ng/mL), l-glutamine (0.3 g/L), and sodium bicarbonate (1.5 g/L). Cells were trypsinized once a week using trypsin-EDTA (Sigma-Aldrich, Inc.) and incubated at 37 °C in a humidified atmosphere containing 5% CO 2 .
5
Establishment of Pancreatic Cell Lines
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Cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Control pancreas cells (hTERT-HPNE (“human pancreatic Nestin-expressing” cells or HPNE; ATCC® CRL-4023™, control pancreatic cells)) and tumour (AsPC-1 (ATCC® CRL-1682™, pancreatic tumour cells), Panc-1 (ATCC #CRL-1469™, Pancreas ductal epithelioid carcinoma), Panc-10.05 (ATCC #CRL-2547™, Pancreatic epithelial adenocarcinoma), MiaPaCa-2 (ATCC#CRL-1420™, Pancreas ductal epithelioid carcinoma), and BxPC-3 (ATCC #CRL-1687™, Pancreatic adenocarcinoma)) were cultured according to the ATCC-suggested protocols. Unless otherwise specified, cells were grown in their defined optimum growth media, as defined by ATCC. For a parallel set of LC50 assays, cells were grown in a minimal media of MEM plus 1% foetal bovine serum (FBS). In miR studies, cells were cultured and treated with NiCl2 (NiCl2; 50 μM), as described in our previously published data [11 (link)].
6
Pancreatic Cancer Cell Lines Cultivation
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The established human PDA cell lines PANC-1, AsPC-1, and BxPc-3 and the human hTERT-HPNE immortalized pancreatic ductal cell line CRL-4023 were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM (18 mmol/L glucose) supplemented with 10% FCS and 5% HEPES or in ATCC complete growth medium, respectively. Gemcitabine-resistant BxPc-3 cells (BxPc-3/GEM) have been described [11 (link)]. Human primary mesenchymal stem cells (MSCs) and the primary PDA cell lines PaCaDD183, PaCaDD159 and PaCaDD135 were isolated and cultured as previously described [11 (link), 47 (link)]. All cells were grown in a humidified incubator at 37°C and 5% CO2 and authenticated throughout the culture by their typical morphology. The established cell lines were recently authenticated by a commercial service (Multiplexion, Heidelberg, Germany). Mycoplasma-negative cultures were ensured through monthly mycoplasma tests.
7
Established PDAC Cell Line Cultivation
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Established human PDAC cell lines, including MIA-PaCa2 (RRID: CVCL_0428), BxPc-3 (RRID: CVCL_0186), PANC-1 (RRID: CVCL_0480), and AsPC-1 (RRID: CVCL_0152), and the non-malignant pancreatic ductal cell line CRL-4023 (RRID: CVCL_C466) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). PDAC cells were cultured at 37 °C in high-glucose DMEM (Sigma, Deisenhoffen, Germany), 10% FBS (Sigma), and 25 mmol/L HEPES (Thermo Fisher, Dreieich, Germany). CRL-4023 cells were cultured in a glucose-free mixture of 75% DMEM, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, and 25% M3 basal medium (Incell Corporation LLC, San Antonio, TX, USA). Monthly testing using PlasmoTest™ (InvivoGen, San Diego, CA, USA) confirmed the absence of mycoplasma contamination in these cell lines. Additionally, all cell lines underwent recent validation through single-nucleotide polymorphism (SNP) analysis conducted by Multiplexion (Heidelberg, Germany).
8
Authenticated Pancreatic Cancer Cell Lines
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AsPC-1, CRL-4023 and PANC-1 pancreatic cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The established cell lines were recently authenticated by a commercial service (Multiplexion, Heidelberg, Germany). The human, primary pancreatic cancer cell line ASANPaCa was kindly provided by Dr. Nathalia Giese and is described [37 ]. To maintain the authenticity of the cell lines, frozen stocks were prepared from initial stocks, and every three months a new frozen stock was used for experiments. Monthly testing ensured mycoplasma negative cultures. Cells were cultured in DMEM (PAA, Pasching, Austria) supplemented with 10% heat-inactivated FCS (Sigma, Deisenhoffen, Germany) and 25 mmol/L HEPES (PAA, Pasching, Austria).
9
Establishment and Characterization of Pancreatic Cancer Cell Lines
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The established human pancreatic cancer cell lines BxPc-3 and AsPC-1 and the human hTERT-HPNE immortalized nonmalignant pancreatic duct cell line CRL-4023 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured as described (55 (link)). Gemcitabine-resistant BxPc-3 cells (BxGEM) were selected from parental cells by continuous gemcitabine treatment with increasing concentrations up to 200 nM for more than one year (56 (link)). Negative mycoplasma cultures were confirmed by monthly mycoplasma tests. The cell lines were recently authenticated by a commercial service (DSMZ, Braunschweig, Germany).
10
Cultivation of PDAC Cell Lines
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The established human PDAC cell lines, MIA-PaCa2 (RRID : CVCL_0428), BxPc-3 (RRID : CVCL_0186), PANC-1 (RRID : CVCL_0480), and AsPC-1 (RRID : CVCL_0152), as well as the nonmalignant pancreatic ductal cell line, CRL-4023 (RRID : CVCL_C466), were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Gemcitabine-resistant BxGEM cells were selected from parental BxPc-3 cells (RRID : CVCL_0186), as described (39 (link)). PDAC cells were cultured at 37°C in high glucose DMEM (Sigma, Deisenhoffen, Germany), 10% FBS (Sigma), and 25 mmol/L HEPES (Thermo Fisher, Dreieich, Germany). CRL-4023 cells were cultured in 75% DMEM without glucose, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, and 25% M3 Base medium (Incell Corporation LLC, San Antonio, TX, USA). Mycoplasma-negative cultures were ensured monthly by PlasmoTest™ (In vivoGen, San Diego, CA, USA). All cell lines have been authenticated by SNP profiling (Multiplexion, Heidelberg, Germany).
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