Calcium Phosphate
Transfection of PC12 Cells
M.
Greenberg lab (617-735-8075)
A.
CELLS:
1. Polylysine coat plates.
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Add 5 ml
polylysine per 100 mm plate
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Put at 37¡C overnight (can also do for 3 h).
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Remove and
save polylysine.
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Wash plates
2X with 10 ml sterile water.
2. Seed cells: 1/2X split from
confluent plate (approximately 2Ð5X, 106 cells per 100 mm plate) in
PC12 culture media.
3. Incubate overnight in 10% CO2
incubator.
4. Cells should be 50Ð70% 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.
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Need 1.4 ml
ppt/100 mm (half is 2X HeBS, half is DNA/CaCl2 mixture).
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20Ð30 ug of
total DNA required per 100 mm plate.
o
5Ð20 ug
reporter plasmid.
o
3Ð4 ug
alpha-globin internal control.
o
pUC19 to
bring to 20Ð30 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 5Ð7 h undisturbed.
C.
GLYCEROL SHOCK:
1. Warm 2X HeBS, PC12 culture medium, PBS, 50% glycerol (all
sterile).
2. Make up 25% glycerol/1X HeBS.
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Will need 2
ml per 100 mm plate.
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1 volume
50% glyc + 1 volume 2X HeBS.
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Typically,
room temp when use.
3. Check plates to confirm normal ppt accumulation.
4. Aspirate off medium (process 4Ð5 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 (37¼C) 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 36Ð48 h, analyze.
1.
Polylysine
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Sigma
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5 mg (one
bottle) in 250 ml sterile H2O (tf 20 ug/ml)
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Store at 4¡C, re-use for a few months
2. PC12
Culture Media
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DMEM (lab
home-made)
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10% horse
serum
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5% fetal
bovine serum [calf serum may also be fine]
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2 mM
glutamine (1/100 from lab stock)
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Penn/strep
(1/200 from lab stock)
3. PC12
Transfection Media
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DMEM
(Gibco/BRL #11960-028; use fairly new bottle so pH is consistent)
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10% calf
serum
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2 mM
glutamine (1/100 from lab stock)
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Penn/strep
(1/200 from lab stock)
o
Make fresh,
day of transfection
4.
Plasmid DNAs
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Purify by
double cesium banding
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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
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Add
components to 1.8 L water
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pH with 10N
NaOH, to pH 7.05
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Bring to 2
L
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Recheck pH;
bring to 7.05Ð7.07
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Filter
sterilize
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Aliquot;
parafilm tubes
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Store at
-20¡C; thaw individual
aliquots as use
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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
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These
recipes are for undifferentiated PC12 cells, in 100 mm plates
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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