Freshly sorted lineage-labelled cells were resuspended in culture medium (3D basic medium (DMEM/F12, Gibco) supplemented with 10% FBS. (Gibco) and ITS (Insulin-Transferrin-Selenium, Corning)), and mixed with cultured lung stromal cells negatively isolated by microbeads of CD326/EpCAM, CD45, and CD31 via MACS (Miltenyi Biotech), followed by resuspension in GFR-Matrigel (BD Biosciences) at a ratio of 1:5. A 100 μl mixture was placed in a 24-well Transwell insert with a 0.4 μm pore (Corning). Approximately 5×103 epithelial cells were seeded in each insert. 500 μl of culture medium was placed in the lower chamber, and medium was changed every other day. ROCK inhibitor Y27632 (10μM, Sigma) was added in the medium for the first 2 days of culture. Analysis of colony forming efficiency and size of organoids was performed at 14 days after plating if there is no specific description.
24 well transwell insert with a 0.4 μm pore
Manufactured by Corning
The 24-well Transwell insert with a 0.4 μm pore is a laboratory equipment product. It is designed to facilitate cell culture experiments and studies by providing a platform for the separation and interaction of different cell types or materials across a permeable membrane.
Automatically generated - may contain errors
Lab products found in correlation
Evos microscope, by Thermo Fisher Scientific (4 mentions)
Insulin transferrin selenium, by Corning (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (3 mentions)
Gfr matrigel, by BD (2 mentions)
Rock inhibitor y 27632, by Merck Group (2 mentions)
Insulin transferrin selenium, by Merck Group (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Growth factor reduced matrigel, by BD (1 mentions)
5 protocols using 24 well transwell insert with a 0.4 μm pore
1
Lung Organoid Formation from Lineage-Labeled Cells
2
Lung Organoid Co-culture Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lung organoid co-culture assays have been previously described46 (link). Lung epithelial cells (Epcam+CD45-CD31-Ter119-) from control BALB/cJ mice or irradiated mice were FACS sorted and resuspended in 3D organoid media (DMEM/F12 with 10% FBS, 100 U/ml penicillin-streptomycin, and insulin/transferrin/selenium (Merck Sigma-Aldrich)). Cells were mixed with murine normal lung fibroblast (MLg) cells and resuspended in GFR Matrigel at a ratio of 1:1. 100μl of this mixture was pipetted into a 24-well transwell insert with a 0.4 μm pore (Corning). 5,000 epithelial cells and 25,000 MLg cells were seeded in each insert. After incubating for 30 min at 37°C, 500μl organoid media was added to the lower chamber and media changed every other day. Bright-field images were acquired after 14 days using an EVOS microscope (ThermoFisher Scientific), and quantified using FiJi (version 2.0.0-rc-69/1.52r, ImageJ).
3
Lung Organoid Formation from Lineage-Labeled Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly sorted lineage-labelled cells were resuspended in culture medium (3D basic medium (DMEM/F12, Gibco) supplemented with 10% FBS. (Gibco) and ITS (Insulin-Transferrin-Selenium, Corning)), and mixed with cultured lung stromal cells negatively isolated by microbeads of CD326/EpCAM, CD45, and CD31 via MACS (Miltenyi Biotech), followed by resuspension in GFR-Matrigel (BD Biosciences) at a ratio of 1:5. A 100 μl mixture was placed in a 24-well Transwell insert with a 0.4 μm pore (Corning). Approximately 5×103 epithelial cells were seeded in each insert. 500 μl of culture medium was placed in the lower chamber, and medium was changed every other day. ROCK inhibitor Y27632 (10μM, Sigma) was added in the medium for the first 2 days of culture. Analysis of colony forming efficiency and size of organoids was performed at 14 days after plating if there is no specific description.
4
3D Co-Culture of Lung Epithelial and Stromal Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly sorted secretory cells were resuspended in culture medium (3D basic medium (DMEM/F12, Gibco) supplemented with 10% FBS (Gibco) and ITS (Insulin‐Transferrin‐Selenium, Corning)). These cells were mixed with cultured lung stromal cells negatively isolated by microbeads of CD326/EpCAM, CD45, and CD31 via MACS (Miltenyi Biotech), followed by resuspension in growth factor‐reduced Matrigel (BD Biosciences) at a ratio of 1:5. A 100 μl mixture was placed in a 24‐well Transwell insert with a 0.4 μm pore (Corning) (Lee et al, 2014 (link)). Approximately 5 × 103 epithelial cells were seeded in each insert. 500 μl of culture medium was placed in the lower chamber, and the medium was changed every other day. For amino acid media change experiment, 500 µl of 3D basic media, 10% dialyzed FBS in BME media (Thermofisher, 21010046), or 10% dialyzed FBS in BME media with EAA (1 mM of L‐Histidine, L‐Isoleucine, L‐Leucine, L‐Methionine, L‐Threonine, L‐Valine, L‐Glutamine, L‐Arginine, L‐Cysteine) was replaced every day.
5
Lung Organoid Co-culture Assay
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!