Calcium Phosphate Transfection of PC12 Cells

 

M. Greenberg lab (617-735-8075)

 

 

I. Protocol

 

A. CELLS:

1. Polylysine coat plates.

       Add 5 ml polylysine per 100 mm plate

       Put at 37C overnight (can also do for 3 h).

       Remove and save polylysine.

       Wash plates 2X with 10 ml sterile water.

2. Seed cells: 1/2X split from confluent plate (approximately 25X, 106 cells per 100 mm plate) in PC12 culture media.

3. Incubate overnight in 10% CO2 incubator.

4. Cells should be 5070% confluent.

5. Remove medium from cells and replace with PC12 transfection media (10 ml/100 mm plate).

6. Place in 5% CO2 to equilibrate, for approximately 1 h (meanwhile, make CaP ppt).

 

B. Ca2PO4 PRECIPITATION:

1. Thaw DNA and 2X HeBS just to room temperature.

2. Calculate amount of DNA and volumes for ppt.

       Need 1.4 ml ppt/100 mm (half is 2X HeBS, half is DNA/CaCl2 mixture).

       2030 ug of total DNA required per 100 mm plate.

o      520 ug reporter plasmid.

o      34 ug alpha-globin internal control.

o      pUC19 to bring to 2030 ug total DNA.

3. Aliquot volume of 2X HeBS to 1 set of tubes.

4. Aliquot sterile water to second set of tubes.

5. To H2O, add the appropriate amount of DNAs.

6. Add 0.l volume (of this half) of 2.5 M CaCl2 (i.e., 70 ul per 100 mm plate worth of ppt).

7. Using a 5 ml pipette, mix DNA/CaCl2.

8. Transfer this dropwise to 2X HeBS, while gently mixing.

9. Let sit 20 min in the dark at end, solution should look slightly cloudy.

10. Add 1.4 ml of ppt dropwise to each plate.

11. Tilt gently to mix

12. Put back in 5% CO2.

13. Incubate 57 h undisturbed.

 

C. GLYCEROL SHOCK:

1. Warm 2X HeBS, PC12 culture medium, PBS, 50% glycerol (all sterile).

2. Make up 25% glycerol/1X HeBS.

       Will need 2 ml per 100 mm plate.

       1 volume 50% glyc + 1 volume 2X HeBS.

       Typically, room temp when use.

3. Check plates to confirm normal ppt accumulation.

4. Aspirate off medium (process 45 plates at a time; e.g., sets of plates to be compared to each other).

5. Start timer (set to 3 min).

6. Add 2 ml glycerol to each plate (start every 15 sec); scatter drops, then stack plates and rock to spread.

7. Let sit 2 min.

8. Under scope cells should appear shrunken.

9. At 2 min aspirate off liquid every 15 sec.

10. Add 4 ml PBS (37C) per 100 mm; aspirate, tilting plates to loosen ppt; wash 2X more with 4 ml PBS/plate.

11. Add 10 ml fresh PC12 culture media; return to 10% CO2 incubator.

12. Repeat for each set of 4 plates.

13. Incubate 3648 h, analyze.

 

II. Reagents

1. Polylysine

       Sigma

       5 mg (one bottle) in 250 ml sterile H2O (tf 20 ug/ml)

       Store at 4C, re-use for a few months

 

2. PC12 Culture Media

       DMEM (lab home-made)

       10% horse serum

       5% fetal bovine serum [calf serum may also be fine]

       2 mM glutamine (1/100 from lab stock)

       Penn/strep (1/200 from lab stock)

 

3. PC12 Transfection Media

       DMEM (Gibco/BRL #11960-028; use fairly new bottle so pH is consistent)

       10% calf serum

       2 mM glutamine (1/100 from lab stock)

       Penn/strep (1/200 from lab stock)

o      Make fresh, day of transfection

 

4. Plasmid DNAs

       Purify by double cesium banding

       For transient transfections, no need to sterilize the DNA by ethanol pptn

 

5. 2X HeBs

Final conc ror 2 liters supplier;

NaCl                               274 mM      32 g       Baker #3624-05; Mallinckrodt

KCl                                  10 mM         1.42 g    Mallinckrodt #6858

Na2HPO4.7H2O           1.4 mM        0.76 g    Mallinckrodt #7914

Dextrose (D-glucose) 15 mM         5.4 g      Baker #1916-01; Mallinckrodt

Hepes (free acid)         42 mM         20 g       Calbiochem #910150

       Add components to 1.8 L water

       pH with 10N NaOH, to pH 7.05

       Bring to 2 L

       Recheck pH; bring to 7.057.07

       Filter sterilize

       Aliquot; parafilm tubes

       Store at -20C; thaw individual aliquots as use

       Note, pH is critical; therefore:

o      Use accurate pH meter

o      Standardize pH meter (with pH 4.0 and 7.0 standards) repeatedly, until standards are read precisely

 

III. Notes

         These recipes are for undifferentiated PC12 cells, in 100 mm plates

         Have also used, per 35 mm plate:

o      1.5 ml transfection media

o      0.3 ml CaP/DNA precipitate

o      4 ug plasmid DNA