Acinar cell 3D culture was performed as previously described (Qu and Konieczny, 2013 (link)). Briefly, pancreta from two 5- to 6-week-old WT and KC littermates were dissociated using 10 mg/ml collagenase P (Sigma-Aldrich, 11213857001) for 20 min at 37°C with agitation. The dissociated pancreta were then washed three times in Hanks’ balanced salt solution (HBSS; Gibco, 14175095) with 5% fetal bovine serum (FBS) before pipetting through a 500-µm (PluriSelect, 352360) and then 100-µm (Corning, 352360) cell sieve. Strained acinar cell clusters were then layered on HBSS+30% FBS and centrifuged at 112 g . The cell pellet was resuspended in 3D culture medium: RPMI (Gibco, 21875-034) containing 50 ng/ml TGFα (Peprotech), 1% FBS, 1% penicillin/streptomycin (Gibco, 15140122), 1 µg/ml dexamethasone (Abcam, ab142419) and either 250 nM MLN8237 (SelleckChem, S1133), 5 µM Tubacin (Stratech, A4501-APE) or DMSO for untreated controls. This was mixed 1:1 with rat tail collagen (Thermo Fisher Scientific, A1048301) and plated in a 24 well plate pre-coated with collagen or a four-well glass-bottom µ-Slide (Ibidi, 80427). Culture medium was added to the wells once set and exchanged 1 and 3 days after plating. Cultures were analysed after 7 days.
Rat tail collagen
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Rat tail collagen is a naturally-derived extracellular matrix protein commonly used in cell culture and tissue engineering applications. It is extracted from the tails of rats and purified to provide a consistent and reliable source of this important biomaterial.
Automatically generated - may contain errors
Lab products found in correlation
Nerve growth factor (ngf), by R&D Systems (2 mentions)
Countess 2 automated cell counter, by Thermo Fisher Scientific (2 mentions)
Transwell inserts 6 well with 8 m pores, by Greiner (2 mentions)
Mln8237, by Selleck Chemicals (1 mentions)
Collagenase p, by Merck Group (1 mentions)
Prism software, by GraphPad (1 mentions)
Dnaase, by Merck Group (1 mentions)
Pronase, by Roche (1 mentions)
Pronase, by Merck Group (1 mentions)
Dmem f12 glutamax medium, by Thermo Fisher Scientific (1 mentions)
Primaria plates, by Avantor (1 mentions)
Bovine pituitary extract, by Thermo Fisher Scientific (1 mentions)
Dnase solution, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Cholera toxin, by Merck Group (1 mentions)
Retinoic acid, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Uridine, by Merck Group (1 mentions)
30 protocols using rat tail collagen
1
3D Acinar Cell Culture Protocol
2
Cell Collision Assay for RNA Scope
3
Quantifying B. subtilis Biofilm Formation
4
Isolation and Culture of Murine Tracheal Epithelial Cells
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Mice tracheas were collected from wild type C57BL/6J (WT). Using pronase (1.5 mg/mL) (Roche®), tracheas were digested overnight at 4°C. Digestion was stopped using MTEC/Basic medium (Composition Table X) + 10% fetal bovine serum (FBS). The DNAase (0.5 mg/mL) (Sigma-Aldrich®) step was realized in MTEC/Basic medium without FBS. Adherent cells were removed by plating samples on specific petri dishes (Primaria Corning). Of the trachea epithelial cells, 33.103 put in 150 µL of MTEC/Plus medium (Composition Table 1
At day 4, the apical medium was changed for MTEC/Plus medium until cell confluence. Basolateral medium was changed every day with MTEC/SF differentiation medium (CompositionTable 1
At day 4, the apical medium was changed for MTEC/Plus medium until cell confluence. Basolateral medium was changed every day with MTEC/SF differentiation medium (Composition
5
Isolating Primary Airway Epithelial Cells
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Trachea from WT mice were collected in DMEM-F12 GlutaMAX medium (Thermofisher Scientific) containing Penicillin-Streptomycin (100 U/mL, Fisher) cocktail and incubated over night at 4°C with Pronase (1.5 mg/mL, Sigma-Aldrich). Cells were collected and washed 3 times with DMEM-F12 GlutaMAX-10% FBS, 100 μm filtered, and centrifuged at 1,500 rpm for 10 min. Cell pellet was resuspended in DNAse solution (0.5 mg/mL, Sigma-Aldrich) for 5 min. Suspension was centrifuged for 10 min at 1,500 rpm and the pellet was resuspended in DMEM-F12 GlutaMAX media and incubated in Primaria plates (353801, VWR) at 37°C for 4 h to remove potential fibroblasts. Unadherent cells were then collected, centrifuged at 1,500 rpm for 10 min, counted and plated in 24 well-plate coated with rat tail collagen (ThermoFisher Scientific) at 50 000 cells per well. At this step, DMEM-F12 GlutaMAX-5% FBS medium containing Insulin-Transferrin (10 μM ThermoFisher Scientific), Cholera Toxin (0.10 μg/mL, Sigma-Aldrich) Epidermal Growth Factor (0.025 μg/mL, Sigma-Aldrich), Bovine Pituitary Extract (30 μg/mL, Fisher), and Retinoic acid (50 nM, Sigma-Aldrich) was used to maintain epithelial cells prior to stimulation. Supernatant and intracellular fraction were stored at −80°C for further analysis.
6
ADM Assay for Pancreatic Duct Formation
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The ADM assay was carried out as previously described7 (link) with slight modifications. Briefly, dissociated primary pancreatic acinar cells were resuspended in DMEM with 1% FBS, 2.5 μM of dexamethasone, and 0.25 mg/mL trypsin inhibitor. This suspension was mixed with a solution containing rat tail collagen (Thermo Fisher Scientific), 10× DMEM, and 0.34 M NaOH. The mixture was poured into 6-well plates, and the collagen gels were allowed to solidify for 30 minutes. Then the enriched cell culture media were added on top of the gel. In certain experiments, the enriched medium was spiked with exogenous IL-6 (PeproTech, Cranbury, NJ) at 2 or 12 ng/mL. The lower concentration of IL-6 was comparable to the concentration of IL-6 detected in the conditioned medium of RAW264.7 cells stimulated with LPS (100 ng/mL) for 6 hours. Enriched media were changed the next day (day 1) and then every 2 days. The total number of ducts per well were counted manually on day 5 using 10× magnification.
7
Measuring Cell Migration and Wound Healing
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Cell migration was measured using a disposable 96 well chemotactic chamber (Neuro Probe, USA). A2780 cells were harvested and suspended in RPMI medium. Membrane filters (8 μm pore size) of the 96 well chemotactic chamber were pre-coated with 20 μg/ml rat-tail collagen (Thermo Fisher Scientific, USA) for 4 h in the clean bench at room temperature. Aliquots (5 × 103/50 μl) of A2780 cells were loaded into the upper chambers of chemotactic chamber, and RPMI with 10% FBS was added into the lower chambers. After incubation at 37°C for 12 h, the membrane filter was washed twice with HBSS and fixed with 4% paraformaldehyde. Migrated cells to the lower surface of each filter were stained with Hoechst dye and then measured by microscopic cell count. For the wound scratch assay, A2780 cells from each condition were scratched with a pipette tip to form a wound. Cells were imaged at 0- and 24-h post-scratching and the distance moved by the cells was measured.
8
DRG Neuron and OPC Co-Culture Protocol
9
Culturing Primary Human Pulmonary Endothelial Cells
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Primary HPAECs were purchased from Lonza (Walkersville, MD). The cells were cultured in standard cell culture T-75 flasks coated with rat tail collagen (R&D Biosystems, Minneapolis, MN). For the cell culture flask coating, the rat tail collagen solution was diluted with sterile cell culturegrade endotoxin-free water (Hyclone, Logan, UT) to a final concentration of 150 µg/mL, which was subsequently added to the flask with sufficient volume to cover the growth surface. The rat tail collagen sorption was incubated at 37 °C and 5% CO 2 for 14-18 h. After sorption, the collagen solution was aspirated and the surface was washed with an equal volume of Dulbecco's phosphate-buffered saline (DPBS) at pH 7.4.
The original HPAECs obtained at passage 3 were cultured and cryopreserved at passage 5 in recover cell freezing media (Thermo Fisher Scientific, Waltham, MA) at a cell density of 1 × 10 6 cells/mL. The cells were cultured in EMB-2 media supplemented with the EGM-2 single-bullet kit (Lonza). For cell detachment from the growth surface, the cell monolayer was washed with prewarmed DPBS at 37 °C without magnesium and calcium and treated with TrypLE solution for 3-4 min at 37 °C and 5% CO 2 . All cellbased assays were performed using cells from passage 5.
The original HPAECs obtained at passage 3 were cultured and cryopreserved at passage 5 in recover cell freezing media (Thermo Fisher Scientific, Waltham, MA) at a cell density of 1 × 10 6 cells/mL. The cells were cultured in EMB-2 media supplemented with the EGM-2 single-bullet kit (Lonza). For cell detachment from the growth surface, the cell monolayer was washed with prewarmed DPBS at 37 °C without magnesium and calcium and treated with TrypLE solution for 3-4 min at 37 °C and 5% CO 2 . All cellbased assays were performed using cells from passage 5.
10
Immunofluorescence Imaging of p50/p105
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The p50/p105-Flp-In cell lines were seeded on coverslips coated with rattail collagen (Thermo Fisher Scientific). Tetracycline-treated cells were induced with TNF (10 or 25 ng/mL for 40 minutes), fixed in paraformaldehyde, and blocked with 1% BSA-PBS (Sigma-Aldrich). The HA.11 antibody was used for detection of tagged p50/p105 with anti-mouse Alexa Fluor 488 (A-11029, Life Technologies) secondary antibody. Nuclei were stained with 49-6-diamidino-2-phenylindole (D9542-5MG, Sigma-Aldrich). Imaging was performed at 340 magnification with the Axio Imager.Z2 (Zeiss, Oberkochen, Germany) and the Nuance multispectral imaging system FX (PerkinElmer, Waltham, Mass). ImageJ software (National Institutes of Health, Bethesda, Md) was used for quantification of signal intensities. 29 Three hundred to 600 cells per condition were analyzed.
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