Neutralized rat tail Collagen I (Fisher Scientific #CB354249) was used to make a thin underlay on 24- or 96-well ultra-low attachment plates (Corning), and allowed to solidify at 37°C until ready for plating. The tissue pellet was resuspended in a mixture of 3:7 Matrigel (Fisher Scientific #08774552) to Collagen I, and the desired amount of matrix/organoid suspension was plated on top of the pre-prepared underlay. Plated organoids were incubated at 37°C, 5% CO2 for 45-60 min. After gelation, pre-warmed AdF+++ supplemented with R-Spondin 3 (250 ng/mL; PeproTech, Rocky Hill, NJ USA), Neuregulin (5 nM; PeproTech), EGF (5 ng/mL PeproTech), Noggin (100 ng/mL; PeproTech), and 1% B27 (Invitrogen) was added to the wells. Sterile PBS was added to empty wells to prevent desiccation. Organoid morphology was examined under phase contrast after 10 d, and the number of branching organoids counted. For qPCR analysis, organoids were collected from Matrigel/Collagen by addition of ice-cold AdF+++ and mixing with Pasteur pipettes. After collection by centrifugation, organoids were resuspended in TriPure Isolation Reagent (Roche Applied Sciences) and processed for qRT-PCR.
24 or 96 well ultra low attachment plates
Manufactured by Corning
Sourced in United States
The 24- or 96-well ultra-low attachment plates are laboratory equipment designed for cell culture applications. These plates feature a surface treatment that minimizes cell attachment, promoting the formation of spheroid and suspension cultures.
Automatically generated - may contain errors
Lab products found in correlation
Neuregulin, by Thermo Fisher Scientific (1 mentions)
R spondin 3, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Noggin, by Thermo Fisher Scientific (1 mentions)
Tripure isolation reagent, by Roche (1 mentions)
Crystal violet dye, by Merck Group (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
2 protocols using 24 or 96 well ultra low attachment plates
1
Organoid Culture Using Collagen I and Matrigel
2
Sphere Formation and Colony Assay
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Cancer cells were trypsinized using 0.25% trypsin and washed twice in PBS in order to prepare single-cell suspensions. Spheroids were photographed after seven days in culture. Two thousand cancer cells per well were seeded into ultra-low-attachment 96-well plates containing 200 μL of the DMEM/F12 supplemented with 5 μg/mL insulin (MilliporeSigma, St. Louis, MO, USA), 20 ng/mL human recombinant epidermal growth factor (EGF; Thermo Fisher Scientific corporation, 10 ng/mL basic fibroblast growth factor (bFGF; Thermo Fisher Scientific corporation, Waltham, MA, USA), and 0.4% bovine serum albumin (BSA; Sigma), followed by culturing in 24- or 96-well ultra low attachment plates (Corning, Corning, NY, USA). Spheroids were photographed after 14 days in culture. For colony formation assays, cells were seeded into 6-cm2 plates at a cell density of 1000 cells per plate. The colonies were counted after staining with crystal violet dye (Sigma) at day 10.
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