Biology Reference
In-Depth Information
A
B
IP
3
100
10 pA
DT40-R3
1s
K
+
DT40-KO
50
DT40-KO
-C
DT40-R3
0
−
10
−
8
−
6
−
4
-C
Log {[IP
3
], M}
Fig. 3
DT40-KO cells provide a null background for expression of functional IP
3
R. (A) IP
3
-evoked
Ca
2
þ
release from permeabilized DT40 cells assessed using a luminal Ca
2
þ
indicator (
Tovey et al., 2006
).
Permeabilized DT40-KO cells stably expressing rat IP
3
R3 (DT40-R3) release Ca
2
þ
when stimulated
with IP
3
, whereas DT40-KO cells are unresponsive. (B) Currents recorded from lumen-out patches from
DT40-KO and DT40-R3 cells. PS included IP
3
(10
m
M), ATP (5 mM), and a free [Ca
2
þ
]of
200 nM;
K
þ
was the charge carrier and the holding potential was
þ
40 mV. C denotes the closed state.
line lacking functional IP
3
R(
Sugawara et al., 1997
). These DT40-KO cells, which
Kurosaki (RIKEN, Japan) has made widely available, provide the only null
background for functional expression of IP
3
R(
Fig. 3
). They have been extensively
used by many groups to express each of the three IP
3
R subtypes and define their
functional properties, and to explore the role of IP
3
R in many higher order
processes (e.g.,
Joseph and Hajnoczky, 2007; Miyakawa et al., 1999
). A recent
review provides further details of the use of DT40 cells for analyses of Ca
2
þ
signaling pathways (
Taylor et al., 2009b
). Here, we describe our use of DT40
cells expressing mammalian IP
3
R for nuclear patch-clamp recording.
IV. Methods
A. Culture of DT40 Cells
Wild-type DT40 cells (Cell Bank number RCB1464) and DT40-KO cells
(RCB1467) are available from Riken Bioresource Center Cell Bank, Japan
(
http://www.brc.riken.jp/lab/cell/
). Cells are grown in RPMI 1640 medium (Invi-
trogen) supplemented with 10% fetal bovine serum (FBS, Sigma), 2 mM
-gluta-
mine, 1% chicken serum (Sigma), and 50
m
M
b
-mercaptoethanol (Invitrogen) in a
humidified atmosphere containing 5% CO
2
, ideally at 39-41
C (matching the
increased body temperature of birds). It is, however, acceptable and more conve-
nient when incubators are shared with mammalian cells to culture DT40 cells at
37
C without loss of viability. The only obvious e
l
ect is a slowing of growth rate;
the doubling time has been reported to increase from about 10 h at 39-41
Cto
18 h at 37
C(
Mak et al., 2006
). The chicken serum must be heat-inactivated
V
