Right now i'm having a problem of sorting viable cells of cultured fetal liver cells. My 90% cells after sorting are non viable.
I'm giving you the protocol that i follow-:
1. Decant the media and wash with PBS twice for 3-4 mins 7-10 ml volume twice.
2. Add 2 ml of trypsin and incubate for 3-4 mins.
3. Add 3-4 ml of FCS containing media and with the help of pipette try to rinse the 90mm for 7-9 times.
4. Add plain media to remove left over adherent cells.
5. Keep these cells in 10% media for 30 mins to revive.
6. Staining with 3% FCS containing media with 100X HEPES kept in Ice, for 30 mins.
7. Wash with 4X the volume of 3% Media.
8. Incubate in secondary for 30 mins in Ice.
9.Wash with the FCS containing media.
10. Resuspend it in 3% media and then sort these cells in 85 micron nozzle.
11. fill the collection tube with media and centrifuge at 1500 rpm for 5 mins.
These cells are bigger in size that i use to analyze at FSC- A of 180-242. They are of fetal stage of 13-15 days old embryo.
Kindly suggest some solution. Hope to listen from you soon.