CaP Transfection of Primary Neurons

Greenberg Lab 1/20/00

I. Protocol

1. Wash plates 3X with DMEM to get rid of serum - save conditioned culture media to return to plates after transfection. Make sure to use only new DMEM that is still orangish - more alkaline. If too pink, the DMEM won’t work. Do not use other base medium even if you normally grow the cells in MEM or Neurobasal.

- transfection media volumes:

well of 24-well plate: 250ul

6 well plate: 2.5 ml

- return plates to 5% CO2 incubator for about 1hour (meanwhile, make precipitates)

2. make ppt.:

a. variables

- volume of ppt

24 well: 12.5µL

6 well plate: 50µL

- amt of DNA :

24 well: 1-4ug

6 well: 2-5µg

b. recipe/procedure for precipitate (to be added to cells):

1) mix DNA + CaCl2 in one 15ml polystyrene pop-cap tube:

1/10 total volume 2.5M CaCl2

DNA

sterile H2O to 500ul

2) aliquot 1/2 total volume 2X HeBS to second tube

3) add DNA/CaCl2 to 2X HeBS dropwise, while swirling 2X HeBS

4) let ppt form in dark, 25’, room temp

3) add ppt to plates: drip over surface

4) leave ppt on cells 20-30 minutes (meanwhile, prepare media)

3. Stop transfection

1) aspirate media, wash 2X DMEM. Leave cells out in hood with second wash for 10-15 minutes to get all of the precipitate up. You may need to increase the length of this step or add more washes especially if the cells are being grown in serum free medium, as if you don’t get all of the precipitate up now, it will continue to accumulate overnight and will kill the cells..

2) add conditioned media - from start of procedure, or other appropriate conditioned media

- to bring cond. media to sufficient volume, add culture media

II. Notes

1. neuronal culture

- this protocol has been used for primary cultures of rat neurons

- for cultures from embryonic animals E17/18, this protocol has worked well for cells from 1-7DIV cortical and hippocampal. Older cells transfect less well. For cerebellar P6 tranfection optimal 4-6DIV.

2. Always co-transfect with GFP (0.1-1µg/well of 24 well) - it gives you a sense of total transfection efficiency quickly and easily.

3. amt of DNA

- 1ug/24-well, 5ug/60mm gave strong signals by Bgal assay or RNAase protection

4. amt of ppt per plate, length of time ppt on plates

- may need to optimize for type, culture age of cells

- check plates periodically; stop if see toxicity

- more ppt seems to form over time, in plates (or, accumulate on plate bottom?); cells seem to tolerate very large amts of ppt (even a “layer” on plate)