PC12 culture and NGF differentiation Z. Xia 5/16/96

Regular PC12 cell Media:

DMEM 475 ml

Horse serum 50 ml (10 %)

Fetal bovine serum(FBS) 25 ml (5 % )

P/S 2.5 ml (from 200X stock/Gibco)

Glutamine (Q) 5 ml

10 % CO2, 37 C

Plates:

Cells are plated onto Collegen coated plates

Collegen coated plates:

1 ml collegen stock (Biomedial Technologies INC, BT-274, keep@ 4 C)

9 ml sterile H2O

mix

Use 0.4 ml/100 mm plate, spread to wet plate surface w/ cell lifter, let dry at 37 C or room temp., use w/i 3 month

To split:

Pipet up-down to trituate cells, split 1:3 or 1:4

NGF differentiation of PC12 cells

Wash cells 2X w/ PBS or serum-free DMEM.

Split cells to 0.9 x 10 ⁶ cells/100 mm plate on collagen coated plates.

10 % CO2, 37 C for 6 days. Then change media every 2-3 days. I usually conduct my experiments 9-14 days after differentiation.

Media for NGF differentiation:

DMEM

1 % Horse serum

P/S

Glutamine (Q)

50 ng/ml NGF (Sigma , recombinant NGF beta, or Austral Biologicals, Cat# GF-022-8, mouse Natural NGF, 500 ug/$300.00, 1-800-433-7105)

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