Calcium
Phosphate Transfection of Neurons in Primary Culture
Developed
by Hank Dudek and Zhengui Xia (11/96)
Modified by
Steve Finkbeiner (rev. 7/97)
Departments
of Neurology and Physiology, UCSF/GIND
415-734-2508;
sfinkbeiner@gladstone.ucsf.edu
I. Protocol
1.
Make calcium phosphate/DNA precipitate:
a. Variables:
§ Volume of ppt
§ 24-well: 20–40 ul
§ 60 mm plate: 120–200 ul
§ Amount of DNA:
24-well:
2–4 ug (e.g., for immunostaining)
60
mm: 2–5 ug (e.g., for RNAase protection)
b.
Recipe: e.g., for 300 ul precipitate (scale up or down accordingly):
§
In a 15 ml polystyrene
pop-cap tube, mix DNA and CaCl2 (1/2 final volume, therefore 150 ul in this
case). 15 ul 2.5M CaCl2 (1/10 of DNA/CaCl2 volume). DNA (e.g., 30 ug = enough
for 10-wells at 30 ul ppt per well) sterile H2O to 150 ul.
§
Aliquot 2X
Hepes-Buffered saline (HeBS) to second tube (150 ul, in this case).
§
Add DNA/CaCl2 to 2X
HeBS dropwise with pipetman, while swirling 2X HeBS.
§
Let ppt form in dark,
25' at room temp.
o
Note: typical plan:
No. wells: 10
Total vol. ppt: 300 ul
DNA/CaCl2 vol.: 150 ul
2.5 M CaCl2: 15 ul
Plasmid DNA: X ul (=30 ug)
Sterile dH2O: X ul (to 150 ul) [add to
tube first]
2X HeBS: 150 ul [make extra volume]
2. Replace culture media with transfection media
(37°C) (near end of 25' ppt formation time).
§ Remove conditioned culture media; save, to return to
plates after transfection.
§ Wash cells 3X with transfection media (serum free,
add, aspirate), then add fresh transfection media (don't let cells dry out).
§ Volume of transfection media:
o Well of 24-well plate: 500 ul
o 35 mm plate: 1.5 ml
o 60 mm plate: 3 ml
3.
Add ppt to plates.
§ Drip evenly over surface, with pipetman.
§ Leave ppt on cells 15–75 min (time varies; see
notes).
§ Leave in air hood if transfection media is MEM, in
incubator if DMEM.
4.
Stop transfection.
§ Aspirate media, wash twice with fresh transfection
media (37°C).
§ Volumes: 24-well: 500 ul; 35 mm: 1 ml; 60 mm: 2 ml.
§ Add back conditioned media.
o Optional: containing 100 uM APV [NMDA receptor
antagonist].
o 24-well: 300 ul; 35 mm: 1.5 ml; 60 mm: 2ml.
o From start of procedure, or other appropriate
conditioned media.
o If necessary, bring conditioned media to sufficient
volume with culture media.
1.
Transfection media: either (see notes):
i. MEM
§ Gibco/BRL# 12370-037 with Hanks' salts with 25 mM
Hepes without L-glutamine.
§ Bring to pH 7.85 with NaOH, sterile filter.
ii.
Or DMEM
§ Gibco/BRL #11960-028.
§ Note: for (i) or (ii): no added serum, penn-strep, or
glutamine.
§ Optional (see notes): include Kynurenate-Mg: 10 vol.
transfection media plus 1 vol. 10X Ky-Mg 10X Ky/Mg stock (10 mM Kynurenic
acid/100 mM MgCl2).
dH2O 170
ml
Kynurenic acid 378
mg
0.5 % Phenol Red 1
ml
1 N NaOH 1.8
ml
1 M Hepes (Sigma) 1
ml
1 M MgCl2 20
ml
o Stir to dissolve, gradually adjusting pH to 7.4 (by
eye) with 1 N NaOH.
o Add dH2O to 200 ml total.
o Sterilize filter (in hood).
o Aliquot to 30 ml; store at -20°C.
o As thaw aliquots, store at 4ºC, up to 1 mo.
Final conc 200
ml Supplier
NaCl 274
mM 3.2
g Baker
#3624-05; Mallinckrodt
KCl 10
mM 142
mg Mallinckrodt
#6858
Na2HPO4.7H2O 1.4 mM 76
mg Mallinckrodt
#7914; 268g/mol
Dextrose (D-glucose) 15
mM 540
mg Baker
#1916-01; 180g/mol
Hepes (free acid) 42
mM 2
g Calbiochem
#391338; 238g/mol
§ Add components to 180 ml water.
§ pH with 5 or 10N NaOH, to pH 7.03.
§ Bring to 200 ml.
§ Bring to pH 7.06 with 1N NaOH; remove portion, save.
§ Bring remainder to pH 7.10; remove portion, save.
§ Bring remainder to pH 7.14.
§ Filter sterilize in cell culture hood.
§ Aliquot to 1 ml aliquots (eppendorf tubes).
§ Store at -20°C; thaw individual aliquots as use.
§ Note, as for any CaP transfection, greatest
efficiency is obtained with a fine, "sandy" ppt. This is critically
dependent on the pH of the HeBS, so it is best to:
o Use an accurate pH meter.
o Standardize the pH meter (with pH 4.0 and 7.0 standards)
repeatedly, until standards are read precisely.
o As indicated above, make and test multiple batches of
2X HeBS, with slightly different pHs (e.g., pH 7.06, 7.10, 7.14); test each,
use best.
o Seems to be good to make fresh 2X HeBS at least every
few months.
1. Amount of
precipitate (ppt) per plate, length of time ppt on plates:
§ The most critical parameters for efficient, nontoxic
calcium phosphate (CaP) transfection of primary neurons are the amount of
CaP/DNA precipitate added to the cells (per volume of media), and the length of
time the precipitate is left on the cells. The length of time the precipitate
is left on is decided based on its efficiency of formation/accumulation; this
is affected by a number of factors including the pH of the HeBS, the quality of
the DNA, and the transfection media composition. The above parameters should
yield a layer of precipitate, starting at 15–30', and becoming heavy by 30–60'.
The neurons tolerate a heavy layer of ppt, and apparently are preferentially
transfected by such a short exposure (15–60 min), compared to glia.
§ With too little ppt, transfection efficiency is low;
with too much, toxicity occurs (either immediate, or with one day delay);
therefore, for a new neuronal cell type, media conditions, or culture age, we
first do a pilot transfection, e.g., with three ppt volumes, and stopping the
transfection after 15, 30, and 60 min. We typically transfect a lacZ expression
vector, followed by immunostaining or X-gal staining (two days later) to assess
transfection efficiency. We achieve approximately 1% transfection efficiency.
§ During the transfection, plates are checked
periodically to assess ppt accumulation and possible toxicity; the transfection
is stopped early if an unusually large amount of ppt has accumulated, or if
there is any toxicity.
§ Usually, a layer of CaP ppt remains on plate even
after washes; this disappears with time, with no apparent ill effect on cells.
2. Transfection
media:
§ In our original protocol (ref. 1), we used DMEM as
the transfection media. The problem with this media is that it is primarily
bicarbonate-buffered, and so the pH can change drastically, and uncontrollably,
during the manipulations of the transfection. The pH of the media in turn
greatly affects the efficiency of precipitate formation (and therefore
transfection efficiency and toxicity). The indicated MEM is Hanks' salts
buffered, and has Hepes, and so maintains its pH in air, allowing consistency
from transfection to transfection. The pH must be raised (here, to 7.85) for
efficient precipitate formation. A drawback with MEM is that the ppt formation
is not as efficient as with DMEM. Therefore, in some cases it may still be best
to use DMEM (e.g., if the pH variability can be controlled, and to achieve
maximum ppt formation).
3. The wash with
transfection media (before placing the cells in transfection media) seems to be
important for removing a residual media component that inhibits precipitate
formation/accumulation. This component may be serum, and therefore the wash may
be unnecessary if cells are grown in serum-free media.
4. Channel
inhibitors:
§ For at least some neuronal types (in particular, more
developed cortical neurons), the inclusion of Kynurenate and APV in the
protocol appears to reduce toxicity. However, these inhibitors are not
essential, particularly if the cells are not very sensitive to excitotoxicity,
and they can also reduce transfection efficiency. Therefore, for new cells, the
transfection can be tried without the inhibitors, or with and without them
side-by-side. For example, we currently do not use Ky or APV for cerebellar
neurons (P6-8 + 6DIV) (note, we haven't yet tried Ky in MEM).
5. Amount of DNA:
§ 2–4 ug/24-well, 5 ug/60 mm gives strong signals by
Bgal staining (X-gal) and RNAase protection, respectively.
§ Supercoiled plasmid DNA, double CsCl-banded, is used.
6. Glycerol
shock:
§ Glycerol shock seemed to only slightly increase
transfection efficiency, and increased damage to cell processes (1 and 5%
glycerol tried).
7. Cell culture:
§ This CaP protocol has been used successfully for
primary cultures of rat neurons from cortex, hippocampus, striatum, spinal
cord, and cerebellum.
§ For cortical cultures from E17/18 fetuses, or
newborns, this protocol has worked well for cells on or after 3 DIV (day of
culture being 0 DIV); if done on 1 or 2 DIV, there can be high toxicity and low
transfection efficiency.
o
Cell density at seeding cortical (E17/18) cerebellar (P6-8)
24-well:
2 X 105 5
X 105 cell/well
60
mm: 3 X 106 5
X 106 cells/plate
§ This protocol has worked for cells grown in a variety
of different culture medias; however, the particular culture conditions can
change the optimal day of transfection.
8. Amount of
CaCl2 in pptn reaction:
§ Lowering the amount of CaCl2 (to 1/2 or 1/4 the
indicated amount) greatly reduced ppt formation efficiency and transfection
efficiency.
References:
1. Xia, Dudek, Miranti, and
Greenberg. (1996) J. Neurosci.
16:5425–5436.
2. Bonni, Ginty, Dudek, and
Greenberg. (1995) Mol. Cell. Neurosci. 6:168–183.
3. Nikolic, Dudek, Kwon,
Ramos, and Tsai. (1996) Genes Dev.
10:816–825.
4. Dudek, Datta,
Franke, Birnbaum, Yao, Cooper, Segal, Kaplan, and Greenberg. (1997) Science 275:661–665.