Seeking advice... One of our facility users is having difficulty sucessfully plating frog adrenocortical cells after cell sorting. Any troubleshooting ideas would be welcomed. Please address replys either to me or to Stacey Shepard (sshepher@students.uiuc.edu). 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 µg/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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