For further insight, the effects of polyI:C, DEP, or rIL-23 treatment were assessed using a mouse lung epithelial cell line. MLE12 cells (SV40-transformed mouse-derived alveolar epithelial cell line; American Type Culture Collection, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle medium:Ham’s F-12 with 2% fetal bovine serum in a humidified atmosphere at 37 °C with 5% CO2; the cells were then stimulated with different doses of polyI:C (0.01 or 50 μg/mL) or different doses of DEPs (0.01 or 0.1 μg/mL), with or without anti-IL-23p19 antibodies (0.05 μg/mL), for 24 h. The effects of different doses of rIL-23 (0.002 and 0.01 μg/mL) on MLE12 cells were also evaluated. Levels of cytosolic IL-23, nuclear IL-33, and cytosolic TSLP were determined using ELISA (Nuclear/Cytosol Fractionation Kit [NE-PER], Pierce Biotechnology, Rockford, IL, USA).
Mle 12 cells
Manufactured by American Type Culture Collection
Sourced in United States
The MLE-12 cells are murine lung epithelial cells derived from the lungs of C57BL/6 mice. These cells are used as a model for studying lung cell biology and function. The MLE-12 cells exhibit characteristics of alveolar type II cells and can be used in various cellular and molecular biology applications.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Icycler, by Bio-Rad (2 mentions)
Plenti6 krasg12v, by Addgene (2 mentions)
Universal mycoplasma detection kit, by American Type Culture Collection (2 mentions)
Polybrene, by Merck Group (2 mentions)
Cc 2561, by Lonza (2 mentions)
Hek293 cell, by American Type Culture Collection (2 mentions)
Beas 2b cell, by American Type Culture Collection (2 mentions)
Raw264.7 cell, by American Type Culture Collection (2 mentions)
C57bl 6 mice, by Charles River Laboratories (2 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions)
Raw264.7 macrophage, by American Type Culture Collection (1 mentions)
Bay11 7082, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Penicillin streptomycin, by Procell (1 mentions)
Amaxa nucleofector kit 5, by Lonza (1 mentions)
Odn2088, by InvivoGen (1 mentions)
49 protocols using mle 12 cells
1
Modeling Lung Epithelial Cell Responses
2
Lung Epithelial Cell Culture and Transfection
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MLE-12 cells, the type II mouse-lung epithelial cell, were purchased from ATCC (Manassas, VA). Cells were cultured in a 50:50 mixed medium of DMEM and Ham’s F-12 supplemented with 4% FBS, insulin (5 μg/mL), transferrin (10 μg/mL), sodium selenite (30 nM), hydrocortisone (10 nM), β-estradiol (10 nM), HEPES (10 nM), and L-glutamine (2 mM). In transgenic studies, MLE-12 cells were cultured on 6-well plates. At 60–75% confluence, transient transfection was carried out using SPAK siRNA (50 nM) (Dharmacon RNA Technologies) as the SPAK-knockdown (SPAK-KD) or siCONTROL Non-Targeting siRNA (50 nM) as the negative control. In the hyperoxic group, cells were placed in an incubator filled with 95% O2 and 5% CO2 at 37 °C for 48 h. In the control group, cells were kept in 21% O2 and 5% CO2 at 37 °C for 48 h.
3
Macrophage Polarization and Lung Epithelial IFN-β Response
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RAW264.7 macrophages (American Type Culture Collection (ATCC), Manassas, VA, USA) were stimulated with IL-4 (10 ng/ml) and IL-13 (10 ng/ml) for 24 h at 37°C and followed by with or without IFN-β stimulation (5, 50 U/ml) for 24 h at 37°C. In some experiments, IL-4/IL-13 stimulated macrophages were treated with AS1517499 (1 μg/ml), which is a STAT6 inhibitor, for 24 h at 37°C. In a separate experiment, MLE-12 cells (ATCC), which are mouse lung epithelial cells, were stimulated with IFN-β (0.01, 0.1, 1 μg/ml) for 24 h at 37°C.
4
Hyperoxia exposure of MLE-12 lung cells
5
MLE-12 Cell Oxygen Exposure Conditions
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MLE‐12 cells (ATCC) were grown in Dulbecco's medium:Ham's F12 (50:50 mix) with 2% FBS, 1% Pen/strep, 10 nM hydrocortisone, insulin‐transferrin‐selenium (0.005 mg/ml insulin, 0.01 mg/ml transferrin, 30 nM sodium selenite), and 10 nM β‐estradiol. Cells at 50% confluency were incubated at 37°C in air (21% O2 and 5% CO2) or hyperoxia (95% O2 and 5% CO2) for 4, 8, 12 or 24 h in a humidified chamber.
6
Hyperoxia exposure of MLE-12 lung cells
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MLE-12 cells were purchased from ATCC. Cells were cultured in DMEM/F12 media containing 2% FBS, insulin (5μg/mL), transferrin (10 μg/mL), sodium selenite (30nM), hydrocortisone (10nM), β-estradiol (10nM), HEPES (10nM) and glutamine (2mM). In air, cells were maintained at 37° C (21% O2, 5% CO2). In the acute model, cells were exposed to 4 hours of hyperoxia (95% O2, 5% CO2). In the recovery model cells were exposed to 4 hours of hyperoxia (95% O2, 5% CO2), then placed in air (21% O2, 5% CO2) for 24 hours.
7
Quantification of Lung Cell Chemokines
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Mouse lung epithelial cells (MLE12 cells) were obtained from ATCC (Manassas, VA, USA). Real-time PCR was performed using an iCycler™ (Bio-Rad, Hercules, California, USA) to determine the levels of CCL17 (thymus and activation-regulated chemokine, TARC), CCL22 (macrophage-derived chemokine, MDC), CCL11 (eotaxin), thymic stromal lymphopoietin (TSLP), and IL-25 mRNA. GAPDH was used as an internal reference. Primers and PCR conditions have been described previously [30] (link)–[32] (link).
8
Culturing MLE-12 Cells in DMEM/F-12
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MLE-12 cells (ATCC CRL-2110), were incubated at 37°C in air containing 5% carbon dioxide and 80-90% humidity. Nutrients were provided in DMEM/F-12, GlutaMAX culture medium containing 2% fetal bovine serum (FBS) solution, and 1% penicillin/streptomycin antibiotics. The culture medium was replaced every two days for cell culture maintenance. All media, supplements, and reactants were purchased from Gibco, ThermoFisher Scientific, Waltham, MA, USA.
9
Characterization of KRAS-driven Cell Lines
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HBEC-3KT (obtained from ATCC) and HBEC3KT-KP cells with KRASG12V expressing (/K) ± p53 shRNA downregulated (/P; a gift from Drs. John Minna and Jerry Shay; ref. 16 (link)) were cultured as described (10 (link)). HBEC3KT-KP cells were authenticated via Western immunoblotting and qPCR for p53 and KRASG12V. MLE12 cells (obtained from ATCC) were cultured as described (17 (link)). These cell lines were transduced with pLEX304-eGFP, –wild-type (WT) C9b, or -AT/GG Mut C9b lentivirus treated with 8 μg/mL polybrene (Sigma) and selected with blasticidin. MLE12/K and HBEC-3KT/K cells are generated by transducing cells with pLenti6-KRASG12V (Addgene). Plate colony formation assays (cell survival) and soft agar colony formation assays (AIG) were undertaken as described (7 (link)). All cell lines were used within 6 passages from receipt and thawing. Cell lines were tested every 2 months for Mycoplasma (Universal Mycoplasma Detection Kit, ATCC) throughout the study starting 2 months after thawing cells received from ATCC and immediately for other cell lines herein described. Parental cell lines were authenticated by short tandem repeat profiling. Cells were treated with Bay 11-7082 (Sigma, 0–10 μmol/L range) at the indicated doses.
10
Isolation and Culture of Mouse Lung Fibroblasts
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Primary mouse lung fibroblasts were obtained using a digestion method. Mice were anesthetized with sodium pentobarbital, and their hearts were lavaged with pre-chilled PBS. Lung tissues were then excised, placed in pre-chilled PBS, and cut into 1–2 cm2 pieces. The lung tissue was digested in DMEM digestion medium containing 1 mg/mL collagenase I at 37 °C for 1 h. Following digestion, cells were passed through 70 and 40 μm cell filters, centrifuged, and resuspended in a DMEM high-glucose medium supplemented with 20% fetal bovine serum (FBS, Sigma-Aldrich) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA). Cells were cultured at 37 °C in a humidified atmosphere with 5% CO2. The identification of primary fibroblasts was confirmed using Vimentin staining (Supplementary Figure S1 ).
MLE12 cells (ATCC, Manassas, VA, USA) were cultured at 37 °C in a humidified atmosphere with 5% CO2 using complete medium containing 10% FBS (Gibco) and 1% penicillin/streptomycin (Procell Life Science & Technology, Wuhan, China). MLE12 cells were seeded into a 12-well plate (1 × 105 cells per well) and cultured for 24 h until the cell density reached 60%. The cells were then stimulated with H2O2 (100 μM, Sigma-Aldrich, USA) for 2 h, followed by washing twice with PBS, and further incubated with fresh medium (with or without CCG) for 72 h.
MLE12 cells (ATCC, Manassas, VA, USA) were cultured at 37 °C in a humidified atmosphere with 5% CO2 using complete medium containing 10% FBS (Gibco) and 1% penicillin/streptomycin (Procell Life Science & Technology, Wuhan, China). MLE12 cells were seeded into a 12-well plate (1 × 105 cells per well) and cultured for 24 h until the cell density reached 60%. The cells were then stimulated with H2O2 (100 μM, Sigma-Aldrich, USA) for 2 h, followed by washing twice with PBS, and further incubated with fresh medium (with or without CCG) for 72 h.
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