Flow cytometry and sorting

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Amitjais
Amitjais's picture
Flow cytometry and sorting

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.

Thanking you

marcus muench
marcus muench's picture
 I'm assuming your trying to

 I'm assuming your trying to sort hepatocytes/hepatoblasts?  If so, these cells are quite delicate.  Try to limit all those washes and reduce the speed of the centrifuge to minimize the g-force.  Plate some unsorted cells to make sure you are actually going to the sorted with viable cells.

Amitjais
Amitjais's picture
i'm looking for some MSCs

i'm looking for some MSCs from these early stage fetal liver. Do i need to follow the same precaution that you had suggested?

marcus muench
marcus muench's picture
 MSC should be much hardier.

 MSC should be much hardier.  You may consider centrifugation on a light-density layer (1.077 g/ml, nycodenz or ficoll etc) to remove dead cells and mature hepatic cells.  In the bone marrow at least, MSC are generally light -density.