For those interested here is a summery of the replies I received concerning the viability of P-19 embryonic carcinoma after sorting. Thanks to everyone, we shall try your suggestions. Ayala 1. Dave Chianese: > Usually post sort cells like LOTS of serum. Go as high as 20 - 30%. Since you > are in small volumes, the cost is not an issue. Be careful not to let them dry > too much. Osmolarity can get too high real fast in multiwell plates. 2. Dr. Douglas E. Swartzendruber: > Increased %FBS, HEPES, feeder cells, low sheath pressure are some basic > approaches. Also, your Ab or ligand used to identify them could be > triggering apoptosis. Try treating (staining) them, washing, then > incubating overnight to check for viability. 3. Dr. Douglas E. Swartzendruber: > Do you know the clonogenicity percentage of the F19's? 100% 10% 1%?? > My experience with EC cells is that they like neighbors; thus, perhaps the > wells could have PYS cells or some similar terato-derived cell line grown to > confluence and then irradiated, to possibly provide sustenance to the singular > EC cells.....just a thought. 4. Rochelle Diamond: > Check your sheath ph prior to sorting and the ph post sorting. We have > found problems depending on the cells. Many cell lines we add hepes > buffer to the pbs to stabilize the ph around 7.3 > Some cells we also gas immediately with CO2 post sort. We put the plates > in a ziplock baggie and add the smae gas that they grow in normally. 5. Simon_Q_Rice: > Have you tried seeding the wells with a feeder layer such as 3T3, or using > conditioned medium in the wells prior to sorting? Its also worth checking > that the cells are viable pre-sorting as well. 6. Dennis J. Young: > Try 10 per well first. If they all grow, then feeder cells will help. But > many may not grow, so you may get clones after all. > Irradiated or mitomycin c treated cells will supply growth factors and will > not proliferate.
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