Biology Reference
In-Depth Information
(56
C for 30 min). We avoid antibiotics, but addition of penicillin (10,000 units/
ml) and streptomycin (10 mg/ml) to culture media is optional (
Winding and
Berchtold, 2001
). The cells grow in suspension in flasks, Petri dishes, or multiwell
plates (Greiner Bio-one). Cells are passaged by 20-fold dilution every 2-3 days
when they reach a density of about 2
10
6
cells/ml. Avoid growing cells beyond a
10
6
ml
1
) in either culture medium or FBS
supplemented with 10% dimethylsulfoxide (DMSO, Sigma) can be frozen and then
stored in liquid nitrogen following standard procedures. We routinely culture cells
for 30-35 passages, before thawing a new frozen stock. For the latter, 1 ml of cells
is dispensed into 20 ml of medium. We find it unnecessary to remove residual
DMSO at this stage. Cells are then passaged after 24 h.
DT40 cells are not easy to transfect with IP
3
R expression constructs, we there-
fore use cell lines stably expressing rat IP
3
R1 (GenBank accession number of
GQ233032.1), mouse IP
3
R2 (AB182290), and rat IP
3
R3 (GQ233031.1). Details
of the methods and sources of the original clones are provided in previous pub-
lications (
Dellis et al., 2006; Rahman et al., 2009; Rossi et al., 2009; Tovey et al.,
2010
). Briefly, DT40-KO cells are transfected by nucleofection with linearized
constructs of pcDNA3.2-IP
3
R using solution T and program B23 (Amaxa) using
5
m
g DNA/10
6
cells. G418 (Invitrogen, 2 mg/ml) is used for selection. Expression
of IP
3
R in each cell line is quantified by immunoblotting using custom-made anti-
peptide antisera (
Cardy et al., 1997; Dellis et al., 2006; Rossi et al., 2009; Tovey
et al., 2010
) and, where needed, by
10
6
cells/ml. Cells (2
density of 2.5
3
H-IP
3
binding. Functional expression of
IP
3
R in each DT40 cell line is verified by comparison with DT40-KO cells using
a luminal Ca
2
þ
indicator and a high-throughput assay for IP
3
-evoked Ca
2
þ
release
(
Laude et al., 2005; Tovey et al., 2006
)(
Fig. 3
). Only cell lines shown to express
functional IP
3
R are used for nuclear patch-clamp recording.
B. Isolation of Nuclei
Several methods have been described for isolation of nuclei; most rely on a
combination of osmotic and mechanical lysis of cells (
Boehning et al., 2001a;
Bustamante, 1994; Franco-Obregon et al., 2000; Marchenko et al., 2005
). Our
protocol is adapted from that of
Boehning et al. (2001a)
. DT40 cells expressing a
recombinant IP
3
R (DT40-IP
3
R cells, 1.5-2
10
6
cells/ml) are centrifuged (500
g
for 2 min at 4
C), washed once with ice-cold phosphate-bu
ered saline (PBS), and
then once with cold nuclear isolation medium (NIM). PBS has the following
composition: 137 mM NaCl, 2.7 mM KCl, 10 mM Na
2
PO4, KH
2
PO
4
, pH 7.4,
with NaOH. NIM comprises: 250 mM sucrose, 150 mM KCl, 3 mM
b
-mercap-
toethanol, 10 mM Tris-HCl, 1 mM phenylmethanesulfonyl fluoride (PMSF,
Sigma), pH 7.5. Cell pellets are resuspended in NIM supplemented with complete
protease inhibitor cocktail (Roche, 1 mini-tablet/20 ml) and stored on ice for up to
4-5 h. For isolation of nuclei, 1 ml of the cell suspension is homogenized with 3-4
strokes of a Dounce homogenizer (Wheaton Industries, Inc.), which lyses about
5-10% of cells, assessed by staining with Trypan Blue (0.001%). This crude lysate
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