I am looking for a protocol for the isolation and in-vitro culture of type I alveolar epithelial cells. Can it be done using sorting of the cells or enzymatic digestion of the lung?
Hi Danielle,
This paper describes the isolation of type I alveolar epithelial cells: Type I Alveolar Epithelial Phenotype in Primary Culture
Shaohua Wang and Rolf D. Hubmayr
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3095986/
Good luck!
I think it is always preferable the Cell Sorting (digestion with collagenase, marking and selection cells in stertility)
After the execution of the sorting and isolation of cell, FITC marker is catabolized in a few hours, usually without damage to crops.
From my Paper in attchment:
1. "Skin samples were digested with 0·1% collagenase type XI (Sigma‐Aldrich) for 2 h at 37 °C. The pellet was washed twice in Dulbecco’s modified Eagle’s medium (DMEM)‐LG (Gibco‐Life Technologies INC.), free of any supplements, centrifuged at 160g for 10 min".
After (remember that every step must be executed always in sterility):
2. Pellet, resuspende in DMEM - High Glucose, without FCS.
3. Markers At this point usually insert anti-CK MAb or MAbs desired already fluoresceinated, then put the whole in an incubator at 37 ° C for 15 minutes.
4. Samples. To centrifuge at 160g for 8 mins, at room temperature.
5. Then, the marked pellet must be resuspended in in PBS at pH 7·4,
6. putting the samples thus obtained into special tubes for Sorter, always in sterility.
7. Prepare the special tubes for cell exit Sorter withe pure FCS, always in sterility.
8. Perform sorting, collecting labeled cells and selected in the tubes with pure FCS. 9. As soon as possible the selected cells must be placed in continue culture at 37°C
10. So immediately resuspend in DMEM-HG the cells, without centrifuging (to avoid a further stress) but only trough diluition.
11. The final cell suspension must be diluited at 1:2 or to the maximum at 1:4 in respect to the origin solution of cells in pure-FCS.
It will take from 2 days to a week before the cells are fully resume. It is recommended not to change or dilute the ground before we see a marked proliferation.
- Badges
- Science topic