MiaPaCa-2 pancreatic cancer cells and L3.6pl metastatic pancreatic cancer cells were grown in monolayers with DMEM medium, supplemented with 10% FBS, 2 mM L-glutamine, penicillin (50 IU/mL), and streptomycin (50 mg/mL). The cells were cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% O2. MiaPaCa-2 cells were purchased from ATCC, Manassas, VA in January 2015, and L3.6pl cells were provided by Dr. Jose Riveno, University of Florida, Gainesville, Florida in 2013. Immortalized human pancreatic normal epithelial (HPNE) cells were obtained from Dr. Paul Campbell, University of North Carolina in 2013 (16 (link)). These cell lines are well characterized and tested at regular intervals using RT-PCR and mycoplasma kit.
Mia paca 2 cell
Manufactured by American Type Culture Collection
Sourced in United States
The MIA PaCa-2 cells are a human pancreatic carcinoma cell line derived from a primary tumor. These cells are adherent and can be used for in vitro studies.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (13 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (9 mentions)
Panc 1 cells, by American Type Culture Collection (5 mentions)
Panc 1, by American Type Culture Collection (5 mentions)
Aspc 1, by American Type Culture Collection (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Hek293t cell, by American Type Culture Collection (3 mentions)
Hek293t, by American Type Culture Collection (3 mentions)
Mcf 7 cell, by American Type Culture Collection (3 mentions)
Hela cell, by American Type Culture Collection (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (2 mentions)
Mycoalert mycoplasma detection kit, by Lonza (2 mentions)
Hek293ft cells, by Thermo Fisher Scientific (2 mentions)
Shh light 2 cells, by American Type Culture Collection (2 mentions)
Victor plate reader, by PerkinElmer (2 mentions)
58 protocols using mia paca 2 cell
1
Cultivation and Characterization of Pancreatic Cancer Cell Lines
2
Cell Culture Protocols for Diverse Cell Lines
3
Isolation and Culture of Cancer-Associated Fibroblasts
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Human CAFs were isolated from the pancreatic cancer tissue of patients, and mouse CAFs were isolated from the tumor tissue of a subcutaneous Panc02 mouse model according to the method of Sharon and colleagues16 (link). Briefly, the tissues were cut into 2- to 3-mm pieces within a small amount of phosphate-buffered saline (PBS) on ice. The small fragments were then dissociated with a collagenase solution. The reaction was stopped by adding cold DMEM + 10% fetal bovine serum (FBS). The single-cell suspension was then used to isolate CAFs by fluorescence-activated cell sorting (FACS). MIA Paca-2 cells were purchased from the ATCC (Manassas, VA, USA). Panc02 cell lines were purchased from the Cell Resource Center of the Chinese Academy of Sciences (Shanghai, P.R. China). MIA Paca-2 cells and CAFs were cultured in DMEM, and Panc02 cells were cultured in RPMI-1640 medium. Both kinds of medium were supplemented with 100 U/ml of penicillin, 100 μg/ml of streptomycin, 2 mM l -glutamine, and 10% heat-inactivated FBS. All cells were cultured within a humidified incubator at 37°C with 5% CO2. Cells were subcultured when they reached 80%–100% confluence.
4
Triptolide and Hypoxia Signaling Modulation
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MIA PaCa-2 cells (ATCC) were cultured in DMEM medium (Hyclone) supplemented with 10% FBS and 1% penicillin streptomycin. S2-VP10 cells were cultured in RPMI 1640 medium (Hyclone) with 10% FBS and 1% penicillin streptomycin. Triptolide was used at a concentration of 50 nM in all in vitro experiments based on the response of this compound from our previous studies18 (link), 20 (link), 21 (link) (unless otherwise mentioned). The hypoxia mimetic cobalt chloride (CoCl2) (Sigma) was used at a concentration of 200 uM for HIF-1α reporter assays. The proteasome inhibitor MG-132 (Sigma) was used at a concentration of 10 uM for HIF-1α reporter assays. BAY87-2243, a hypoxia inhibitor was used in indicated concentration. Minnelide, the pro drug of triptolide, was used in vivo at indicated concentrations.
5
Lentiviral Transduction of MIA PaCa-2 Cells
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HEK293T cells (ATCC, Manassas, VA) and MIA PaCa-2 cells (human
pancreatic cancer; ATCC, Manassas, VA) were maintained in Dulbecco’s
modified Eagle’s medium (Corning, Lowell, MA) supplemented
with 10% fetal bovine serum (Corning, Lowell, MA) and penicillin–streptomycin
(Corning, Lowell, MA). All cells were grown at 37 °C and 5% CO2 in a humidified incubator and tested negative for mycoplasma
using the MycoAlert Mycoplasma Detection Kit (LT07-318, Lonza, Morristown,
NJ).
Lentivirus was produced by transfection of HEK293T packaging
cells with three packaging plasmids30 (link) (pMDL,
pRSV-REV, and pCMV-VSVG) and the lentiviral reporter plasmid using
Lipofectamine 3000 (Invitrogen, Waltham, MA) according to the manufacturer’s
recommended protocol. The supernatant containing lentivirus was then
collected 72 h later, aliquoted and stored at −80 °C.
MIA PaCa-2 cells were seeded on a 6-well plate (Corning, Glendale,
AZ) at a density of 3 × 105 cells/well and transduced
with 500 μL lentiviral supernatant plus polybrene (MilliporeSigma,
Burlington, MA) to a final concentration of 8 μg/mL. Transduced
cells were then expanded further in culture and FACS sorted for mStrawberry
expression (BD-FACS Aria, BD Bioscience, Franklin Lakes, NJ).
pancreatic cancer; ATCC, Manassas, VA) were maintained in Dulbecco’s
modified Eagle’s medium (Corning, Lowell, MA) supplemented
with 10% fetal bovine serum (Corning, Lowell, MA) and penicillin–streptomycin
(Corning, Lowell, MA). All cells were grown at 37 °C and 5% CO2 in a humidified incubator and tested negative for mycoplasma
using the MycoAlert Mycoplasma Detection Kit (LT07-318, Lonza, Morristown,
NJ).
Lentivirus was produced by transfection of HEK293T packaging
cells with three packaging plasmids30 (link) (pMDL,
pRSV-REV, and pCMV-VSVG) and the lentiviral reporter plasmid using
Lipofectamine 3000 (Invitrogen, Waltham, MA) according to the manufacturer’s
recommended protocol. The supernatant containing lentivirus was then
collected 72 h later, aliquoted and stored at −80 °C.
MIA PaCa-2 cells were seeded on a 6-well plate (Corning, Glendale,
AZ) at a density of 3 × 105 cells/well and transduced
with 500 μL lentiviral supernatant plus polybrene (MilliporeSigma,
Burlington, MA) to a final concentration of 8 μg/mL. Transduced
cells were then expanded further in culture and FACS sorted for mStrawberry
expression (BD-FACS Aria, BD Bioscience, Franklin Lakes, NJ).
6
Establishment of Mouse Pancreatic Cancer Cell Lines
7
Cell Culture Protocols for Cancer Cell Lines
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HEK293FT cells (ThermoFisher #70,007), MIA PaCa-2 cells (ATCC #CRM-CRL-1420), and Panc-1 (ATCC #CRL-1469) were cultured in D10 media: DMEM (Lonza #12-614Q) supplemented with 10% FBS, and 0.5% Penicillin-Streptomycin. All cell lines were maintained at 37 °C and 5% CO2. Cells were cryopreserved with the addition of 10% DMSO (EMD #MX1458-6) to D10 media.
8
Cell Culture Conditions for Cancer and Immune Cells
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MiaPaCa-2 cells (ATCC CRL-1420) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (Pen-Strep). Materials are provided in the Supplementary Information. MCF-7 cells (ATCC HTB-22) were cultured in DMEM/F12 supplemented with 10% FBS, 0.01 mg/mL insulin, 2 mM Glutamax, and 1% Pen-Strep. MDA-MB-231 (ATCC HTB-26) cells were cultured in DMEM (high glucose) supplemented with 10% FBS and 1% Pen-Strep. NK92-CD16 cells (ATCC PTA-6967) were cultured in Prime XV NK Cell Chemically Defined Medium (Irvine Scientific, Irvine, CA) supplemented with 100 U/mL of IL-2 (PeproTech, Cranbury, NJ) and 1% Pen-Strep. The cancer cells were grown in T75 flasks and NK cells were grown in 24-well and 6-well ultra-low attachment plates. The cells were all maintained in a 37 °C incubator at 5% CO2. The cancer cells were passaged when they reached 70–80% confluent. The NK cells were passaged when they reached 1 × 106 cells/mL and the medium was exchanged every 2 days.
9
Shh Signaling Pathway Modulation in HEK 293T and MIA PaCa-2 Cells
10
Pancreatic Cancer Spheroid Formation
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Human PDAC PANC−1 cells (ATCC-CRL−1469), human pancreas adenocarcinoma ascites metastasis cells AsPC−1 (ATCC-CRL−1682), and undifferentiated human pancreatic carcinoma MIAPaCa-2 cells (ATCC-CRL−1420) were purchased from ATCC (LGC Standard s.r.l., Sesto San Giovanni (MI), Italy), and were cultured as previously reported [42 (link)]. Human pancreatic cancer stllate cancer-associated fibroblasts (CAF08, PC00B5) were purchased from Vitro Biopharma (Golden, Colorado). All materials for cell culturing were purchased from EuroClone (Pero, Milan, Italy).
For PANC−1 tumor homo-spheroids formation, cells were seeded at a density of 5000 cells per well on 96-well Corning Spheroid Microplate (Corning, NY, USA) and incubated at 37 °C under 5% CO2 for 5 days before performing experiments. The spheroids of PANC−1 in co-culture with CAF cells were obtained, as described above for homospheroids using a 2:1 PANC−1:CAF ratio. Spheroid formation was assessed using OLYMPUS CKX41 microscope (OLYMPUS, Tokyo, Japan).
For PANC−1 tumor homo-spheroids formation, cells were seeded at a density of 5000 cells per well on 96-well Corning Spheroid Microplate (Corning, NY, USA) and incubated at 37 °C under 5% CO2 for 5 days before performing experiments. The spheroids of PANC−1 in co-culture with CAF cells were obtained, as described above for homospheroids using a 2:1 PANC−1:CAF ratio. Spheroid formation was assessed using OLYMPUS CKX41 microscope (OLYMPUS, Tokyo, Japan).
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