Transfection of NGF-differentiated PC12 cells
Zhengui Xia, 12/13/96
Prepare glass coverslips
1. Shake coverslips in many changes of ddH2O, including one overnight shake.
2. With coverslips spread evenly on the bottom of the flask or baking dish, autoclave with the dry cycle. Let dry.
3. Place 5Ð6 15 mm coverslips in each 35 mm dish. Coat with poly-D-lysine and laminin. (Dilute 1 100 ul aliquot of 1 mg/ml laminin and 1 100 ul aliquot of 0.1 mg/ml PDL in 20 ml water.)
1. Differentiate PC12 cells in differentiation medium (DMEM with 1% horse serum and 100 ng/ml NGF, see differentiation protocol) 7Ð10 days.
2. Two days before transfection, split to 0.85x 106 cells per 35 mm dish with coverslips. Use 2Ð3 ml differentiation medium per dish.
3. Optional: the day before transfection, change medium to reduce toxicity.
For each 35 mm plate,
1. Mix 2 ug DNA and 100 ul OptiMEM.
2. In separate tube, mix 15 ul Lipofectamine and 100 ul OptiMEM.
3. Mix the two solutions together. Keep at RT 45Õ.
4. Mix in 0.8 ml DMEM (Gibco/BRL #11960-010) supplemented with 2 mM Q but without pen-strep.
5. Wash plates once with 2 ml DMEM + Q without pen-strep.
6. Incubate in the 1 ml transfection medium 37ûC, 10% CO2, 5 h.
7. Wash plates with 2 ml differentiation medium. Grow cells in differentiation medium.
8. To reduce toxicity change the medium every day.
9. Harvest cells 48Ð72 h after transfection. (To study NGF deprivation-induced apoptosis, harvest cells 15Ð19 h after NGF deprivation.)