Human normal pancreatic duct cell line hTERT-HPNE (CRL-4023; ATCC, Manassas, Virginia, USA) was grown in Dulbecco’s modified Eagle’s medium (E600010; Sangon Biotech, Shanghai, China) with 5% fetal bovine serum (FBS, C0234; Beyotime, Shanghai, China), 10 ng/mL recombinant human EGF (P5552; Beyotime), 5.5 mM d -glucose (G7021; Sigma-Aldrich), and 750 ng/mL puromycin (ST551; Beyotime) as recommended by the producer. PCa cell lines BxPC3 (C1023), SW1990 (C1196), and PANC1 (C1004) were available from WHELAB (Shanghai, China). BxPC3 cells were maintained in RPMI-1640 medium (E600028; Sangon Biotech), SW1990 cells were cultured in F-12 basic medium (M0500; WHELAB), and PANC1 cells (C1004; WHELAB) were grown in the high-glucose DMEM (M0100; WHELAB). All media for PCa cells were blended with 10% FBS and 1% penicillin–streptomycin (G0100; WHELAB). Cell incubation was completed in an incubator (HF100; Heal Force, Shanghai, China) at 37°C with 5% CO2.
Htert hpne
Manufactured by American Type Culture Collection
Sourced in United States, China
The HTERT-HPNE is a cell line derived from human pancreatic duct epithelial cells. It is immortalized through the introduction of the human telomerase reverse transcriptase (HTERT) gene.
Automatically generated - may contain errors
Lab products found in correlation
Panc 1, by American Type Culture Collection (63 mentions)
Bxpc 3, by American Type Culture Collection (44 mentions)
Mia paca 2, by American Type Culture Collection (40 mentions)
Aspc 1, by American Type Culture Collection (34 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (26 mentions)
Capan 2, by American Type Culture Collection (23 mentions)
Cfpac 1, by American Type Culture Collection (20 mentions)
Sw1990, by American Type Culture Collection (18 mentions)
Capan 1, by American Type Culture Collection (16 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (16 mentions)
Medium m3 base, by INCELL (11 mentions)
Htert hpne, by Thermo Fisher Scientific (11 mentions)
Panc 1, by Thermo Fisher Scientific (8 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (8 mentions)
Rpmi 1640, by Thermo Fisher Scientific (8 mentions)
Bxpc 3, by Thermo Fisher Scientific (7 mentions)
Aspc 1, by Thermo Fisher Scientific (7 mentions)
Hpaf 2, by American Type Culture Collection (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Mia paca 2, by Thermo Fisher Scientific (5 mentions)
95 protocols using htert hpne
1
Culture and Maintenance of Pancreatic Cell Lines
2
Culturing Pancreatic Cancer Cell Lines
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The human pancreatic cancer cell lines Mia PaCa-2, BxPC-3, Panc-1, human pancreatic cells UACC-462 and hTERT-HPNE were purchased from ATCC (Manassas, VA, USA) and cultured based on the ATCC established guidelines. Human KP3 was obtained from JCRB cell bank (Tokyo, Japan) while murine pancreatic adenocarcinoma cell line Panc-2 was obtained from Frederick National Laboratory for Cancer Research (Frederick, MD, USA). All pancreatic cancer cells were either cultured in DMEM or RPMI1640 medium supplemented with 10% of fetal bovine serum (FBS) and 1% of penicillin/streptomycin. UACC-462 was cultured in Leibovitz’s L-15 medium supplemented with 5% of FBS. hTERT-HPNE cells were cultured with a mixture of 75% DMEM (no glucose; Thermo Fisher Scientific, Waltham, MA, USA) and 25% Medium M3 Base (InCell, Frisco, TX, USA) supplemented with 5% FBS, 10 ng/mL human recombinant epidermal growth factor, 1 g/L glucose, and 750 ng/mL puromycin. All cells were maintained in 37°C humidified incubator supplemented with 5% CO2.
3
Culturing Pancreatic Cancer Cell Lines
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CD18/HPAF, Capan-1 and MiaPaCa-1 pancreatic cancer cells and human immortalized pancreatic ductal epithelial cells hTERT-HPNE were purchased from ATCC and were maintained as per the ATCC recommendations and based on our previous publications [11 (link), 12 (link), 14 (link), 45 (link)]. The pan EGFR family members specific and EGFR specific inhibitors used in this study (Selleck Chemicals, TX, USA) were dissolved in either DMSO (canertinib and erlotinib) or in PBS (afatinib) and stored at −20°C as per the manufacturer's recommendation.
4
Culturing Pancreatic Cancer and Nestin-Expressing Cell Lines
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Pancreatic cancer cell-line PANC-1 was procured from ATCC (CRL-1469; Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (30–2002, ATCC) supplemented with 10% FBS (Sigma, St.Louis, MI, USA). MIA PaCa-2 was obtained from ATCC (CRM-CRL-1420) and cultured in Dulbecco’s Modified Eagle’s Medium (30–2002, ATCC) supplemented with 10% FBS. AsPC-1 was obtained from ATCC (CRL-1682), and cultured in an RPMI-1640 medium (30–2001, ATCC) supplemented with 10% FBS (Sigma). Capan-1 was obtained from ATCC (HTB-79), and cultured in an Iscove’s Modified Dulbecco’s Medium (30–2005, ATCC) supplemented with 20% FBS (Sigma).
A human pancreatic nestin-expressing cell line,27 (link) hTERT-HPNE, was obtained from ATCC and cultured in amixture of 75% DMEM without glucose (Cat#. D-5030; Sigma, with additional 2 mM L-glutamine and 1.5 g/L sodium bicarbonate) and 25% Medium M3 Base (Cat# M300F-500; Incell Corp., San Antonio, TX, USA) supplemented with 10% FBS (Sigma). All the cells were cultured at 37°C in 5% CO2.
A human pancreatic nestin-expressing cell line,27 (link) hTERT-HPNE, was obtained from ATCC and cultured in amixture of 75% DMEM without glucose (Cat#. D-5030; Sigma, with additional 2 mM L-glutamine and 1.5 g/L sodium bicarbonate) and 25% Medium M3 Base (Cat# M300F-500; Incell Corp., San Antonio, TX, USA) supplemented with 10% FBS (Sigma). All the cells were cultured at 37°C in 5% CO2.
5
Maintenance of Pancreatic Cell Lines
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Pancreatic cell lines hTERT-HPNE (ATCC® CRL-4023™), Capan-2 (ATCC® HTB-80™) and AsPC-1 (ATCC® CRL-1682™) were obtained from ATCC (Manassas, VA, USA). Each cell line was maintained in specialized medium according to the supplier’s specifications.
6
Diverse Pancreatic Cancer Cell Lines
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The BxPC-3 (CRL-1687), PANC-1 (CRL-1469), MIA Paca-2 (CRL-1420), and AsPC-1 (CRL-1682) cell lines were obtained from ATCC. Additionally, the PK45P (RCB2141), PK45H (RCB1973), PK8 (RCB2700), PK59 (RCB1901), and KP-4 (RCB1005) cell lines were purchased from RIKEN Cell Bank (RCB), and maintained under the same conditions as PDAC cell lines. The control hTERT-immortalized pancreatic epithelial cell line (hTert HPNE) (15 (link)) was obtained from ATCC.
7
Culturing Pancreatic Cell Lines
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Normal pancrease cell line hTERT-HPNE and pancreatic cancer cell lines BxPC-3, Capan-1, Capan-2, HPAC, MIA PaCa-2, Panc 02.03, Panc 02.13 and PANC-1 were from ATCC (Manassas, VA, USA). The cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.
8
In Vitro Compound Cytotoxicity Screening
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LNCaP (ATCC, Manassas, VA, CRL-1740), DU 145 cells (ATCC, HTB-81), NIH3T3 (ATCC, CRL-1658), hTERT-HPNE (ATCC, CRL-4023), PANC-1 (ATCC, CRL-1469), and 293T (ATCC, CRL-3216) were cultured as recommended by ATCC. Cell lines were authenticated by IDEXX Laboratories in August 2014. A patient-derived fibroblast cell line [36 (link)], which was cultured in RPMI 1640 supplemented with 10% fetal bovine serum, was also tested. Compounds were tested in triplicate. Cells were plated at 4 × 103 cells per well in 96-well plates with 100 μL per well RPMI or DMEM containing 10% v/v fetal bovine serum and 100 μg/mL each of penicillin and streptomycin. Each well was immediately treated during cell plating with one compound at final concentrations of 200 to 0.390625 μM at twofold serial dilutions. Each experiment also included a condition with 0.5% v/v DMSO but no compound. Cell growth was measured using PrestoBlue Cell Viability Reagent (Life Technologies, Carlsbad, CA) following 72 hours of incubation at 37°C in 5% CO2. Eleven microliters of the reagent was added to cells in each well and then incubated 60 minutes at 37°C in 5% CO2. Emission at 612 nm (excitation: 525 nm) was recorded to measure resazurin production. Emission data were normalized so that cells tested against DMSO only (no compound) represent 100% growth.
9
Culturing Cancer Cell Lines
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The cells were cultured at 5% CO2 and 95% of Atmospheric air at 37°C. The cell lines hTERT-HPNE (ATCC® CRL-4023™, Manassas, VA), BxPC3 (ATCC® CRL-1687™, Manassas, VA) and PANC-1 (ATCC® CRL-1469™, Manassas, VA) were cultured in the conditions recommended by the supplier. All the cell lines were used between passages 2–14.
10
Establishing Cas9 Stable Cell Lines
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All cell lines were maintained in a humidified incubator at 37 °C in 5% CO2. PANC-1, hTert HPNE, Mia PaCa-2, HPAF-II, and AsPC-1 cells were purchased from ATCC and used experimentally within five passages. All cell lines were maintained according to ATCC recommendations, and ATCC authenticated the cell lines by short tandem repeat (STR) DNA profiling. The cells were verified to be mycoplasma-free by using the MycoProbe Mycoplasma Detection kit (R&D Systems, Minneapolis, MN). Cas9 stable cell lines were made by virally transducing cells with LentiCAS9-Blast (Addgene, Cambridge, MA; cat. # 52962) [15 (link)] and selecting with 8 μg/mL of blasticidin for 5 days. Expression was verified by Western blot analysis (Additional file 1 b).
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