Confluence & Differentiation in PC-12 Cultures - How is Confluence in PC-12's Determined? Differentiation? (May/03/2007 )

Hi all, I began working with PC-12's this week. This is the first suspension cell culture cell line I've ever worked with so I have a few questions. First, how is confluence in suspension cultures determined? Also, how should they be split? That is to say what ratio is best for PC-12's; I've read no more than 1:3 and at least 1:3 but no more than 1:6 from another source. Or, should seeding be done by number of cells?

In regards to differentiating PC-12's, I'm planning on doing ICC on these cultures but I need them to be differentiated. Should I plate them onto a collagen coated surface and then begin differentiation or should I differentiate in suspension and then plate them just before I need them? Finally, how long does the differentiation process take for PC-12's?

Thanks in advance for any advice and please let me know if you have any questions, comments, or concerns, or if my post isn't clear on the information I need.

Mondo

Update if anyone is interested; I spoke to a professor in my department who did his graduate work with PC-12's and he informed me that these cells could be considered fully differentiated after one week of NGF treatment.

Hi, I plate my cells on Primaria dishes all the time (so not just for differentiation). When I split, I usually do about 1:6.

As for differentiation: one week at low serum and 50 ng/ml NGF should be fine.