Transfection of NGF-differentiated PC12 cells--转染NGF诱导分化的PC12细胞

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 5-6 15mm coverslips in each 35mm dish. Coat with poly-D-lysine and laminin. (Dilute 1 100ul aliquot of 1mg/ml laminin and 1 100ul aliquot of 0.1mg/ml PDL in 20ml water.)

    

Prepare cells

1. Differentiate PC12 cells in differentiation medium (DMEM with 1% horse serum and 100ng/ml NGF, see differentiation protocol) 7-10 days.

    

2. Two days before transfection, split to 0.85x106 cells per 35mm dish with coverslips. Use 2-3ml differentiation medium per dish.

    

3. Optional: the day before transfection, change medium to reduce toxicity.

    

Transfect cells

1. Mix 2ug DNA and 100ul OptiMEM.

    

2. In separate tube, mix 15ul Lipofectamine and 100ul OptiMEM.

    

3. Mix the two solutions together. Keep at RT 45’.

    

4. Mix in 0.8ml DMEM (GibcoBRL 11960-010) supplemented with 2mM Q but without penstrep.

    

5. Wash plates once with 2ml DMEM Q without penstrep.

    

6. Incubate in the 1ml transfection medium 37_C, 10% CO2, 5h.

    

7. Wash plates with 2ml differentiation medium. Grow cells in differentiation medium.

    

8. To reduce toxicity, change the medium every day.

    

9. Harvest cells 48-72h after transfection. (To study NGF deprivation-induced apoptosis, harvest cells 15-19h after NGF deprivation.)