The Protocol I am following is as follows :
1. Isolate the Proximal and cecal part of the colon in ice cold DPBS.
2. Flush the colon using cold DPBS to remove the stool.
3. Put the colon on a wet towel and cut Longitudinally using sterile scissors.
4. Wash the opened colon with DPBS containing Gentamicin (50ug/ml) and Pen strep (100U/ml) for 10 min by gentle shaking with blunt forceps.
5.Transfer to Ice cold DBPS and shake gently with forceps. Now, put the opened colon on a wet towel and remove the mucus gently using rubber scraper.
6. After mucus removal, put the opened colon on petri plate on ice and add 30mM EDTA 1ml per colon and incubate for 30 minutes.
7. Remove the cells using a sterile rubber scraper and immediately add sterile 10 ml PBS and pipette up and down for single cell suspension.
8. Collect the suspension and centrifuge at 500rcf for 10 min at 4C.
9. wash thrice with ice cold DPBS.
10. Wash thrice with FBS free media.
11. Check the viability using Trypan blue and then proceed for plating in a collagen coated plate.
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