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We have successfully developed co-cultures of frog chromaffin and
adrenocortical cells to study cellular interactions between the two cell
types. It is now
desirable to have cultures of each cell type. We are attempting to sort the
cells using flow cytometry by labeling the lipophilic dye Nile Red to
identify lipid-rich adrenocortical cells. However, following the sorting and
plating of cells, very few cells survive and proliferate. The cells appear
to be healthy and intact but they will not plate down. We have tested cells
that have not been sorted but have been stained with the nile red and they
plate down and survive as expected. We have also taken cells left in the
sample tube after a sort and they plate ok as well.
The cell suspension prepared above is sorted using the lipophilic dye Nile
Red to identify adrenocortical cells. Normal frog Ringer's solution is the
sheath solution and cells are sorted into supplemented L-15 medium plus 10%
fetal calf serum. Following the sorting, the cells are centrifuged (300xg,
10 minutes) and resuspended in supplemented L-15 medium plus 5% fetal calf
serum (FCS), nerve growth factor (50 ng/ml) and bFGF (10 ng/ml).The cells
are plated on poly D-lysine (0.1 mg/ml) and laminin (10 =B5g/ml)
substrates.
Thanks,
Stacie L. Shepherd
Neuroscience Program
Medical Scholars Program
University of Illinois at Urbana-Champaign
Gary Durack
University of Illinois Biotechnology Center
voice (217) 244-0559 fax (217) 244-0466
durack@uiuc.edu
http://www.life.uiuc.edu/biotech
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web