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Growth conditions.
We grow our PC12 cells on standard tissue culture plastic without the addition of collagen or poly -L-lysine, in 10% FCS, 5% horse serum, DMEM, 100ug/ml penicillin & streptomycin. Cells are passaged by squirting them off the plastic without prior trypsinisation. They are fairly tightly adherant and so it is best to maintain the stock in flasks where it is easiest to give them a good "blast". The cells normally have a well defined round appearance. Only the occasional cell (a few % or less) should appear differentiated (flattened plus or minus neurite outgrowth). It is probably best to have a large number of vials from a proven passage frozen down as some workers believe that multiple passaging may select for varients. However, bear in mind this line has been passaged many times since its isolation (Greene and Tischler, 1976 PNAS 73 2424-2428).
Vectors.
For transfection we use vectors that utilize the CMV promoter to drive the gene of interest and SV40 early promoter to express neo (pcDNA1 or pcDNA3, Invitrogen). Muller et al. (1990, DNA and Cell Biology 9, 221-229) describe a comparison of vectors using the CMV immediate early promoter, b-actin promoter and MoMuLV LTR to drive expression of CAT and reported that the CMV promoter was better than b-actin and much better than MoMuLV (little or no expression). They also describe a comparison of CaPO4 and lipofectin for transfection of the cells. (Lipofectin being 100-fold better than CaPO4).
Transfection.
We have transfected PC12 cells using both CaPO4 and lipofectamine (Gibco-BRL, #8324SA) and find lipofectamine 100+ better than CaPO4. The protocol is essentially the same as a standard lipofectamine proceedure:
1. D=1 Cells are plated at 1-2 x 105 per 30 mm dish (usually in 6 well costars). DNA is linearized o/n and then phenol extracted, precipitated and resuspended in sterile TE at 1ug/ul.
2. D=2 A transfection complex is prepared:
tube A 100ul DMEM (no FCS) or OptiMEM (Gibco-BRL) plus 2ug DNA
tube B 90ul DMEM (no FCS) or OptiMEM plus 10ul lipofectamine and mix.
3. Gently add contents of A to B and allow to form a precipitate for 20-30min at RT
4. Aspriate medium from the cells and rinse twice with DMEM (no FCS) or OptiMEM.
5. Add 800ul DMEM (no FCS) or OptiMEM to the complex and add to the rinsed cells.
6. Incubate cells for 6hrs then add 1ml DMEM with 20% FCS, 10% horse serum and incubate overnight.
7. D=3 Harvest cells by squirting them off the dish and add 1/5th and 4/5ths to two 10cm dishes respectively, with 10ml media.
8. D=4 Replace media with media containing 400ug/ml G418 (effective concentration) or other appropriate antibiotic. Feed cells twice a week for the first 3 weeks then once a week as the non-transfected cells have died off. Be careful not to dislodge cells during the refeeds.
9. Colonies appear around 3-4 weeks and are usually picked around 5-6 weeks by placing a cloning ring over the top and pipetting the cells off with a p200 Gilson pipette. We generally obtain 50-200 colonies per transfection with p-cDNA based vectors. Cells are removed to 24 well costar plates (15mm) with 1.5ml of media. They are often a bit clumy at this point but can be dispersersed more completely by squirting over the next few days. We keep to antibiotic selection up till freezing. The cells are density dependant and so split them into larger flasks when they are reasonably dense (25-50% confluent in their current container).
Differentiation with NGF. 1. To differentiate the cells we use Nerve Growth Factor (NGF, Sigma 2.5S #N-6009, reconstituted in PBS with 1% FCS and frozen at -70¡C at 25ng/ul). The recomended concentration is 100ng/ml. We find the PC12 cells will differentiate in concentrations as low as 10ng/ml but that the rate is less than at 100ng/ml. At 100ng/ml cell begin to flatten out after 24-26 hrs, after 2d neurite sprouts appear and around 4-5 days there are long neutite processes.