The MLg2908 mouse lung fibroblast cell line CCL-206 (American Type Culture Collection) was cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc.)_ with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). In vitro organoid culture of Club cells in Matrigel has been described previously (19 (link)). In brief, fractionated Club cells were mixed with MLg cells in Matrigel basic medium (1:1). The culture medium included DMEM/F12 (Gibco; Thermo Fisher Scientific, Inc.), insulin/transferrin/selenium (Invitrogen; Thermo Fisher Scientific, Inc.), 10% fetal bovine serum, 100 IU/ml penicillin, 100 µg/ml streptomycin and 10 µM SB431542. The mixture was then loaded into Transwell filter inserts (Greiner BioOne), which were placed in 24-well plates containing the culture medium and cultured in a humidified atmosphere with 5% CO2 at 37˚C. Resulting organoids were visualized using a Zeiss Axiovert40 inverted fluorescent microscope (Zeiss AG). To inhibit the Gabrp-mediated pathway, the cell culture medium was supplemented with 20 µg/ml of BMI (20-23 (link)), and co-cultured for 10 days.
Transwell filter inserts
Manufactured by Greiner
Sourced in Austria
Transwell filter inserts are cell culture inserts designed to create a barrier between two compartments, allowing for the study of cell migration, permeability, and transport processes. The inserts are composed of a microporous membrane that permits the passage of small molecules and ions while retaining cells in the upper chamber.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Ccl 206, by American Type Culture Collection (1 mentions)
Insulin transferrin selenium, by Merck Group (1 mentions)
Sb431542, by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
Dmem f12, by Corning (1 mentions)
Ix73 inverted fluorescence microscope, by Olympus (1 mentions)
2 protocols using transwell filter inserts
1
In vitro Organoid Culture of Club Cells with Lung Fibroblasts
2
Organoid Culture of Alveolar Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sorted club cells (5 × 103 cells/well) or AT2 cells (2 × 104 cells/well) were mixed with MLg fibroblasts in Matrigel (BD Pharmingen, San Diego, Calif)/basic medium (1:1) that contained DMEM/F12 (Corning, China), 10% FBS, 1% insulin-transferrin-selenium (ITS) (Sigma-Aldrich), 100 IU/mL penicillin, 100 µg/mL streptomycin and SB431542 (Sigma-Aldrich). The cell mixtures were then added to Transwell filter inserts (Greiner Bio-One, Kremsmunster, Austria) in 24-well plates containing 410 µL medium. Organoid cultures were maintained in an incubator with 5% CO2 at 37 °C, and the medium was renewed every other day. Organoid cultures were observed through an IX73 inverted fluorescence microscope (Olympus, Tokyo, Japan). Clones with diameters greater than 50 μm were counted, and colony-forming efficiency (CFE) was measured by counting the number of colonies in each well as a proportion of the implanted cells 10 days after seeding. Organoid cultures were embedded in Tissue-Ted optimal cutting temperature (O.C.T.) compound (Sakura, Torrance, Calif) for immunofluorescence or lysed for RNA analyses.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!