Hello every one,
I wants to separate, purify and culture the mice uterus epithelial cells and there are various protocol available, kindly if any one worked in this area suggest me the standard protocol with highest purity and viability of epithelial cells and secondly in case if we used magnetics beads or any other linker or antigen for the separation of epithelial cells then how to detach them from the epithelial cells at the end after getting the epithelial cells?
Your quick and kind response will be highly appreciated
Dissect out the uteri in a dish, flush each uterus with PBS to get rid of existing secretions from the uterus. Tie the uterus on one end with a sterile suture. Fill the uterus with Medium 199+0.2% collagenase + 0.01% deoxyribonuclease from the second end, and tie the second end of uterus with sterile suture. Leave uterus at 37C in the medium (Medium 199, 10%FBS+1%Pen/strep) in the dish or tube with gentle shaking. After 30 min, take out the contents from uterus by flushing with excess of medium (medium 199 and 10%FBS and 1%Pan/step). Wash the cells with medium and than culture them. You should have endometrial epithelial cells in the medium.
Subhash C. Juneja thank you very much Sir for your detailed and well explained answer and as i am new in this field of uterus epithelial cells isolation and purification so i will be very thankful if you shared your published protocol if possible and secondly after getting the endometrial epithelial cells how we have to conform that there are only pure epithelial cells without any other cells type or contaminations? Once again thank you for your time.
With best regards
I used as such. But this suspension may have slight percentage of fibroblast cells. Those are easy to remove by differential plating. You grow the whole suspension in culture, fibroblast cells attach to surface within one hour or so, than you can transfer rest of media in another dish, do it 2/3 times, you will get rid of fibroblasts, rest will be epithelial cells.
If you want to characterize further, you can search markers for epithelial cells and talk to a company. I have not done that myself.
Good luck