Journal: Journal of visualized experiments : JoVE

Article Title: Generation of Organoids from Mouse Extrahepatic Bile Ducts

doi: 10.3791/59544

Figure Lengend Snippet: Materials

Article Snippet: Prepare seeding medium and washing buffer ( ) in 50 mL conical tubes and keep them at 4 °C or on ice until use. table ft1 table-wrap mode="anchored" t5 Name Company Catalog Number Comments L-WRN cell culture medium Advanced DMEM/F12 Life Technologies 12634–010 Fetal Bovine Serum (FBS) 1% Life Technologies 10437–028 Penicillin-Streptomycin 100 U/mL Life Technologies 15140–122 Washing buffer Phosphate Buffered Saline (PBS) 50 mL Life Technologies 10010–023 Penicillin-Streptomycin 125 U/mL Life Technologies 15140–122 Amphotericin B 6.25 μg/mL Life Technologies 15290–018 Organoid culture medium L-WRN Conditioned medium 1:1 ATCC CRL-3276 Advanced DMEM/F12 1:1 Life Technologies 12634–010 Penicillin-Streptomycin 100 U/mL Life Technologies 15140–122 N-Glutamine 10 μl/mL Life Technologies 35050–061 N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, HEPES 10 mM Life Technologies 15630–080 B27 10 μl/mL Gibco 17504–044 N2 10 μl/mL Gibco 17502–048 Organoid seeding medium Organoid culture medium Epidermal growth factor (EGF) 50 ng/mL Invitrogen PMG8041 Fibroblast growth factor-10 (FGF10) 100 ng/mL PeproTech 100–26 Primary antibodies Anti-Cytokeratin 19 (CK19) antibody, Rabbit 1:250 Abcam ab53119 Sex-Determining Region Y-Box 9 (SOX9) antibody, Rabbit 1:200 Santa Cruz sc-20095 Pancreatic Duodenal Homeobox 1 (PDX1) antibody, Rabbit 1:2000 DSRB F109-D12 E-cadherin antibody, Goat 1:500 Santa Cruz sc-31020 Acetylated α-tubulin antibody, Mouse 1:500 Sigma-Aldrich T6793 Secondary antibodies 488 labeled anti-rabbit, Donkey IgG 1:1000 Invitrogen A-21206 594 labeled anti-goat, Donkey IgG 1:1000 Invitrogen A-11058 568 labeled anti-mouse, Goat IgG2b 1:500 Invitrogen A-21144 TopFlash Wnt reporter assay TopFlash HEK293 cell line ATCC CRL-1573 Luciferase Assay Kit Biotium 30003–2 0.05% Trypsin-EDTA Life Technologies 25300054 0.4% Trypan Blue Solution Life Technologies 15250061 Additional materials and reagents Basement matrix, phenol free Matrigel CORNING 356237 Dissociation buffer, Accutase Gibco A1110501 Cell culture freezing medium, Recovery Life Technologies 12648010 Cell strainer (70 μm, steriled) Fisherbrand 22363548 Guanidinium thiocyanate-phenol RNA extraction, TRIzol Invitrogen 15596026 Specimen processing gel, HistoGel Thermo Fisher Scientific HG-4000–012 Universal mycoplasma detection kit ATCC 30–1012K 1.5 mL microcentrifuge tube Fisherbrand 05–408–129 24 well plate USA Scientific CC7682–7524 50 mL conical centrifuge tube Fisher scientific 14–432–22 Fluorescence microscope Nikon Eclipse E800 Inverted microscope Biotium 30003–2 Necropsy tray Fisherbrand 13–814–61 Open in a separate window Materials Set up a surgical table ( ).

Techniques: Cell Culture, Labeling, Reporter Assay, Luciferase, RNA Extraction, Fluorescence, Microscopy, Inverted Microscopy