Mouse lung epithelial cells (MLE12) were obtained from ATCC (Manassas, VA, USA); 3 × 105 MLE12 cells were plated in 24-well plates and incubated overnight at 37°C. The cells were stimulated with rAc-PF and Aspergillus proteins for 3 h. Next, MLE12 cells were collected with 1 mL of QIAzol (Qiagen, Hilden, Germany), and RNA extraction was conducted in accordance with the manufacturer’s protocols for transcription of 2 μg of RNA. Real-time PCR was performed to determine the levels of macrophage-derived chemokine (MDC; CCL22), eotaxin (CCL11), thymic stromal lymphopoietin (TSLP), and IL-25 RNA. GAPDH was used as an internal reference. The primers and PCR conditions used have been described previously [25 (link), 26 (link)].
Mle 12
Manufactured by American Type Culture Collection
Sourced in United States
The MLE-12 is a laboratory instrument designed for the cultivation and maintenance of mammalian cell lines. It provides a controlled environment for cell culture, including temperature, humidity, and gas composition regulation. The MLE-12 is a versatile tool used in various research and clinical applications involving the propagation and study of mammalian cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (18 mentions)
Dmem f12, by Thermo Fisher Scientific (7 mentions)
Mle 12, by Thermo Fisher Scientific (7 mentions)
β estradiol, by Merck Group (6 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Raw264, by American Type Culture Collection (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Beas 2b, by American Type Culture Collection (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Dmem medium, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Mitotracker, by Thermo Fisher Scientific (2 mentions)
Atcc ccl 185, by American Type Culture Collection (2 mentions)
Saecs, by Lonza (2 mentions)
Nih3t3, by American Type Culture Collection (2 mentions)
Hydrocortisone, by Merck Group (2 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
91 protocols using mle 12
1
Transcriptional Response of Lung Epithelial Cells to Aspergillus Proteins
2
Culturing Type II Alveolar Cells
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Type II alveolar epithelial cell lines (MLE-12) were purchased from ATCC (Manassas, VA, USA) and cultured as described previously7 (link). These were routinely maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% foetal bovine serum, 2 mM of l -glutamine, 100 IU/mL of penicillin, and 100 μg/ML of streptomycin (purchased from Invitrogen, Carlsbad, CA) at 37 °C and with 5% CO2 in the air.
3
Isolation and Culture of Murine AECII
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AECII-like MLE12 (mouse) and A549 (human) cells, kidney-epithelial HEK293T cells, and murine fibroblast cell line Mlg were all obtained from ATCC. The isolation of primary murine AECII from the lungs of C57BL/6J mice including their culture is outlined in detail in Online Resource 1. The animal experimental procedures for the isolation of primary AECII from lungs of C57BL/6J were approved by the institutional animal welfare representative and the local governmental ethics committee for animal welfare (Regierungspräsidium Giessen, Germany).All cell culture experiments, including the generation of stably transfected epithelial cell lines MLE12/pBI-L-CHOP and MLE12/pBI-L-EV (empty vector control), are provided in Online Resource 1.
4
Cell Culture and Transfection of Lung Cancer Cell Lines
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Mouse lung epithelial cells MLE-12 (ATCC CRL-2110) were cultured in complete DMEM/F12 (5% FCS, 1% Penn-strep) at 37 °C in 5% CO2. Human SCLC cells NCI-H82 (ATCC HTB-175) and NCI-H196 (ATCC CRL-5823), and NSCLC cells A549 (ATCC CCL-185) were cultured in complete RPMI (10% FCS, 1% Penn-strep) at 37 °C in 5% CO2. During subculturing, cells were 1x PBS washed, trypsinized with 0.25% (w/v) Trypsin and subcultivated at the ratio of 1:5 to 1:10. The cell lines used in this paper were mycoplasma free. They were regularly tested for mycoplasma contamination. In addition, they are not listed in the database of commonly misidentified cell lines maintained by ICLAC. Cells were transfected with plasmid DNA or siRNA using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions, and harvested 48 h later for further analysis. ITGA2-HIS (Addgene, #51910), ITGB2-YFP (Addgene, #8638) and ITGB6-GFP (Addgene, #13293) mammalian expression constructs were used for respective gene overexpression in cell lines. siITGB2 (EHU133911) was purchased from Sigma. Empty vectors and siCtrl were used as a negative control.
5
WT and IL-6 Knockout Mouse Lung Cell Study
6
Cell Line Culture and Differentiation Protocol
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MLE-12, 293T, J774a.1, U937 and A549 cell lines were purchased from ATCC (Manassas, VA). MLE-12 were cultured in HITES medium (50:50, DMEM-Ham’s F-12) supplemented with 2% FBS, 1:100 insulin/transferrin/selenium supplement. 293T and J774a.1 was maintained with high glycose DMEM with 10% FBS, while U937 and A549 were maintained in 1640 medium with 10% FBS. All the cells culture medium was supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml). Before the transfection, U937 was differentiated into macrophage-like cells by 72h stimulation with 200nM PMA (phorbol 12-myristate 13-acetate). HITES medium were purchased from Hyclone (Logan, UT, USA), while DMEM and 1640 medium were provided by Gibco (Grand Island, NY, USA). ITS supplement 100X, penicillin/streptomycin 100X and PMA were supplied by Sigma (St. Louis, MO, USA).
7
Isolation and Culture of Alveolar Macrophages
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MH-S (ATCC CRL-2019) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT) and 100 U/ml of penicillin/streptomycin (P/S, Life Technologies, Rockville, MD) antibiotics in a 37°C incubator with 5% CO2. Mouse AM were isolated by bronchoalveolar lavage (BAL) [46 (link)]. In brief, trachea was cannulated with a 20-gauge catheter; 0.9 ml BAL buffer was instilled, flushed four times, and retrieved. A total of 3.0 ml BALF was retrieved from each mouse and cytospin slides prepared with 0.5 ml BALF were stained by HEMA-3 (Fisher, Rockford, IL) to enumerate leukocyte subtypes based on their cellular and nuclear morphological properties. After centrifugation at 2, 000 rpm, AM cells were resuspended and cultured in RPMI 1640 medium as above. MLE-12 (ATCC CRL-211) were cultured in HITES medium as above. Stable TLR2 expression HEK293 cells (ATCC CRL-157) using a pUNO-TLR2 plasmid were obtained from InvivoGen (San Diego, CA) and cultured in DMEM medium as above.
8
Mitochondrial Regulation in Lung Cell Response
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Human small airway epithelial cells (SAECs) were
purchased from Lonza, Allendale, NJ (LOT: 000470903). Primary mouse AECIIs were
isolated from the lungs of unchallenged mice. Mouse lung epithelial cell line
(MLE12) was purchased from ATCC (CRL-2110). Cells were seeded into 96-well
plates (40.000/well), exposed to bleomycin (15 mU/ml) or PBS for 4 hours and
then treated with T3 (15 ng/ml) or vehicle control for 8 hours.
Immunofluorescence staining was performed using Mito-Tracker (Thermo Fisher
Scientific, Waltham, MA), a cationic dye that stains active mitochondria and
PPARGC1A (ab54481, Abcam, Cambridge, UK), according to manufacturer’s
instructions. Detection of apoptotic cells was performed with TUNEL-assay using
the in situ Cell Death Detection Kit, Fluorescein (Roche, Indianapolis, IA, USA,
Catalog No 11684795910). DAPI staining was used to determine the number of
nuclei and to assess gross cell morphology. The human lung adenocarcinoma cell
line A549 (ATCC® CCL-185™, ATCC, Manassas, VA),
negatively tested for mycoplasma, was cultured in DMEM supplemented with
10% fetal bovine serum. Cells were pre-incubated with either dronedarone
10 μM diluted in 0.04% DMSO or vehicle control (0.04%
DMSO) for 24 hours and then treated with T3 (15 ng/ml) or vehicle (normal saline
0.9%) for 24 hrs.
purchased from Lonza, Allendale, NJ (LOT: 000470903). Primary mouse AECIIs were
isolated from the lungs of unchallenged mice. Mouse lung epithelial cell line
(MLE12) was purchased from ATCC (CRL-2110). Cells were seeded into 96-well
plates (40.000/well), exposed to bleomycin (15 mU/ml) or PBS for 4 hours and
then treated with T3 (15 ng/ml) or vehicle control for 8 hours.
Immunofluorescence staining was performed using Mito-Tracker (Thermo Fisher
Scientific, Waltham, MA), a cationic dye that stains active mitochondria and
PPARGC1A (ab54481, Abcam, Cambridge, UK), according to manufacturer’s
instructions. Detection of apoptotic cells was performed with TUNEL-assay using
the in situ Cell Death Detection Kit, Fluorescein (Roche, Indianapolis, IA, USA,
Catalog No 11684795910). DAPI staining was used to determine the number of
nuclei and to assess gross cell morphology. The human lung adenocarcinoma cell
line A549 (ATCC® CCL-185™, ATCC, Manassas, VA),
negatively tested for mycoplasma, was cultured in DMEM supplemented with
10% fetal bovine serum. Cells were pre-incubated with either dronedarone
10 μM diluted in 0.04% DMSO or vehicle control (0.04%
DMSO) for 24 hours and then treated with T3 (15 ng/ml) or vehicle (normal saline
0.9%) for 24 hrs.
9
Western Blot Analysis of TRPV4 in Lung
10
Culturing Murine Pulmonary Epithelial Cells
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The mouse pulmonary epithelial cell lines MLE-12 and A549 were obtained from ATCC (VA, USA) and cultured in DMEM containing 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin in a 5% CO2 atmosphere at 37°C. MLE-12 or A549 cells were seeded in 6-well plates or 24-well plates overnight and then treated with HDM extract or the cytokine IL-33 for the indicated durations. Primary alveolar epithelial cells from mice were purified using 0.1% collagenase, 0.25% trypsin, and DNase I and were selected with mouse IgG (36111ES60, Yeasen, China) as previously described (27 (link)). To exclude the potential effects of lipopolysaccharide (LPS) contamination, HDM extract was treated with the ToxinEraserTM endotoxin removal Kit (L00338, Genscript, China). The purified product was the major constituent of HDM.
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