PC12 cell protocols

Growing:

--PC12 cells grow in DMEM-Hi supplemented with 15% FBS. They should be plated on collagen-coated plates.

--Media should be changed every 2-3 days. When doing this, remove 75% of the old media and add new. Media should be room temp or 37^o C for feeding.

--Cells can be split 1:5, but no harder. Cells do not require trypsin for passage; merely suck up media and use it to wash cells off of the plate. When splitting, keep at least 20% old media, as they like conditioned media.

Differentiation:

--PC12 cells should be 50-70% confluent for differentiation.

--Differentiate in DMEM-Hi with 15% FBS supplemented with 50 ng/ml NGF.

--Change media to new NGF-containing media every 2-3 days, as above

--Differentiation is complete after 5-7 days

Freezing:

--One 10 cm dish 70-90% confluent will provide 2 vials of frozen cells.

--Dislodge cells from dish(es) as above, collect by low speed centrifugation, and freeze in DMEM-Hi with 20% FBS and 10% DMSO.

--Place in -70^o overnight and then move to LN₂.

Collagen-Coating Plates:

--Dilute Collagen (Biomedical Technologies, Inc. rat tail collagen BT-274) 1:5 in sterile PBS. Add 400 ml of dilute collagen per 10 cm dish in hood, spread with sterile spreader. Allow to dry in hood overnight. Plates are good for about 2-3 weeks.