Cytell - GEヘルスケア・ジャパン株式会社 ライフサイエンス統括本部

imagination at work
ウェスタンブロッティングだけでは見られない
タンパク質発現の局在を定量しませんか
撮影から解析
グラフ化まで1台で！
Cytell Cell Imageing System
660 万円
薬剤・作用溶量・曝露・作用時間などの影響による局在変化をみたい
論文掲載にむけた n=3 の実験で増大するサンプル数の対策に
ウェル毎の平均値だけでなく、特定の細胞集団割合を解析したい
（例：GFP 陽性細胞率）
という方におすすめ
ウェスタンブロッティング
イメージングサイトメーター
細胞培養
細胞培養
35 mm ディッシュ以上の大きなディッ
35/60 mm ディッシュは
シュで培養。
もちろんのこと、
6 ∼ 96
サンプル数分の枚数が必要。 ウェルプレート1 枚での
35/60 mm ディッシュはもちろん
のこと、
6 ∼ 96 ウェルプレート1 枚
Cytell
でのアッセイも可能。
アッセイも可能。
タンパク質抽出
免疫染色
（抗体反応）
電気泳動
細胞の撮影
転 写
解 析
抗体反応
バンドの検出
ウェスタンを
補完する技術
発現量比も
数値化できる！
解 析
コントロール
さらに
X線フィルム画像や
ゲル画像
細胞内の
発現部位が見える
画像解析装置や
表計算ソフトウェアを
用いた数値化
複数のタンパク質
の発現が
同時に見える
取扱店
www.gelifesciences.co.jp
サンプル
Case その
1
細胞内でのタンパク質の発現量と存在パターンを調べたい
細胞内でのタンパク質の発現量・存在パターン
（凝集、散在）
の評価が可能
ウェスタンブロッティングではとらえることが難しい、
タンパク質の存在パターンの変化を、Cytell で解析した例をご紹介します。
FYVE は PI3Kinase によってリン酸化されるドメインの 1 つで、リン酸化されるとエンドソームに凝集します。その性質を利用し、
EGFP 融合 FYVE を安定的に発現させた U-2 OS 細胞で、リン酸化阻害剤（Wortmannin）のさまざまな濃度における発現量および
存在パターンを調べました。
1
2
3
4
5
6
7
8
9
10
11
12
10 µM
5 µM
2.5 µM
1.25 µM
625 nM
312.5 nM
156.3 nM
78.1 nM
39 nM
19.5 nM
9.8 nM
0M
＜プレートフォーマット＞
A
B
C
Plate 1
D
Plate 2
E
Plate 3
F
0M
G
H
※ プレート内の数字は Wortmannin 濃度
1 Cytell で撮影した細胞画像
2 1 細胞あたりのタンパク質発現量
下図 B、 D よりリン酸化阻害剤濃度が高くなることで、FYVE の
凝集が減少し、散在することがわかりました。
1 細胞あたりのタンパク質発現量が確認できるのもイメージング
Hoechst（核）
GFP（2xFYVE）
B
1 細胞あたりの GFP 総蛍光輝度
（RFU x µM2）
2,500,000
リン酸化阻害剤濃度
+0.0195 µM
A
サイトメーターの特長です。リン酸化阻害剤の濃度が変わっても、
発現量に変化がないことがわかりました。
C
D
2,000,000
1,500,000
Plate 1
Plate 2
1,000,000
Plate 3
500,000
0
0.0195
1.25
+1.25 µM
リン酸化阻害剤濃度
（µM）
3 1 細胞あたりのスポット数
1 で撮影した画像から、1 細胞あたりのスポット数を自動的にカ
ウントします。その結果、濃度が高くなるにつれスポット数が減少
することがわかりました。
35.000
FYVE
1 細胞あたりの EGFP-2x
FYVE スポット数
30.000
リン酸化
阻害剤濃度
リン酸化阻害剤の濃度依存的なタンパク質存在パターンの変化
25.000
20.000
Plate 1
15.000
Plate 2
Plate 3
10.000
リン酸化阻害剤濃度
（µM）
2
5
10
2.5
1.25
0.625
0.3125
0.1563
0.039
0.0781
0.0195
0
0.000
0.0098
5.000
Case その
2
シグナルトランダクションや細胞分化、
マーカー発現の解析をしたい
PDGF 刺激によって核移行したリン酸化 ERK の定量解析
x10レンズ
（一部拡大）
PDGF 刺激
PDGF 刺激＋阻害剤
0 min
蛍光シグナルの経時的変化
0 min
10 min
PDGF刺激
340
320
300
280
260
240
220
200
0
10 min
核内シグナル強度
細胞質内シグナル強度
10
20
30
40
50
60
70
80
90
100
PDGF刺激＋阻害剤
340
320
核内シグナル強度
300
細胞質内シグナル強度
280
260
240
220
0
ADCC アッセイ
x10レンズ
抗体
エフェクター細胞
ー
＋
10
20
30
40
50
60
70
80
＋
＋
抗体－
添加直後
抗体＋
1.2
総細胞蛍光シグナル強度
1
添加
6 時間後
0.8
0.6
0.4
0.2
0
添加直後
添加 6 時間後
ターゲット細胞の Calcein 蛍光イメージ
マウス腎臓組織切片のマーカー発現解析
2 色のマーカーによる 2D プロット
Phalloidin 蛍光輝度
x4レンズ
青：DAPI
緑：Alexa Fluor 488 WGA
赤：Alexa Fluor 568 Phalloidin
WGA 蛍光輝度
神経突起伸長解析
x10レンズ
未処理細胞
レチノイン酸処理細胞
3
1 細胞あたりの神経突起長の平均値
N2a 細胞を 96 ウェルプレートに播種し、列ごとにさまざまな濃度のレチノイン酸で処理しました。
レチノイン酸濃度
（µM）
90
100
Pie Chart
transfe
Negative
Positive
Classes
Transf
s
of dete
Cell Count
effect
Cell CountsiGLO
2313
493
1 had
2313 these
493
Transf
favour
of siRN
Transfection Conditions
1. A549 cells were plated in 96-well plates at 10,000 cells/well in medium containing Ham’s F-12, 2 mM L-Glutamine, 1.5 g/L Sodium Bicarbonate and 10% FBS,
of red
24 hr before transfection.
favour
2. siGLO Transfection Indicators and DharmaFECT 1 Transfection Reagent were mixed in reduced serum medium and incubated for 20 min at room temperature.
3. Complete growth medium was added to each siGLO/DharmaFECT 1 mixture, and growth medium on the cells replaced with 100 µL of resulting transfection
transfe
mix. Transfected cells were incubated for 24 hr in 37C 5% CO humidified incubator, fixed with 4% paraformaldehyde, washed with DPBS, and stained with
increa
Hoechst 33342 (1 µM).
B
wells is
siGLO
Red
Imaging and Analysis with Cytell
B
siGLO
1. A 2-color BioApp was created using MyBioApp by setting the Blue Channel to image nuclei (Hoechst) and either the Orange or Green Channel to image
Heat Map
siGLO Red
＊
siGLO fluorescence.
with re
蛍光標識
の
を用いてトランスフェクションの可否を確認した例をご紹介します。Measurement: Heat Map
2. Nuclear reference segmentation, which overlaps the nuclear area, was chosen for the siGLO Transfection Indicators since positive signal will be localized
added
to the nuclear area of each cell.
Measurement:
%
of
Cells
＊ は細胞導入後に核に移行する性質をもつため、
細胞導入の確認が容易となります。ターゲットsiRNA と共にトランスフェクションして使用できます。
siGLO
3. Four
fields were
captured from each well of the 96-well plate.
siRNAs
% of Cells
Population:
4. After imaging and initial analysis were complete, the threshold (linear gate) setting for positive and negative transfection was set immediately upstream
assays
of the strong signal observed from a histogram chart from a well containing a significant positive signal.
Population:
Negative
Positivechart
Case その
3
siRNA のノックダウン効率を調べたい siRNA のトランスフェクションをスムーズに確認できるツールが揃っています
2
siRNA
siGLO Red Transfection Indicator
Positive
90ウェルプレート1 枚でトランスフェクション効率と
Determining optimal siGLO and DharmaFECT 1 amounts for efficient transfection of A549 cells:
プレートマット状の結果から作図も楽々
A two-dimensional dose-response experiment was set up in a 96-well plate to determine efficient transfection conditions for A549 cells (Figure 1). A range of increasing
amounts of siGLO Transfection Indicator (12.5-100 nM) and DharmaFECT 1 (0-0.4 µL/well) were used across the plate as indicated. A549 cells were plated and grown for
24 hr before the addition of the siGLO/DharmaFECT 1 mixture. After an additional 24 hr cells were fixed, stained and analysed on the Cytell Cell Imaging System.
Positive
Sum
細胞増殖を同時に評価
The Cy
and ea
efficien
Indica
Map a
the am
begin
to gen
confid
not ha
Cytell C
Transf
and ea
when
B
50
50
50
50
50
50
50
50
50
50
50
50
nM siGLO RNA
1 Conditions
2
3
4
5
6
7
8
9
10
11
12
Transfection
µL/well
プレートマップ状に数値結果が配置された状態
0
0.1
0.1
0.1
0.15
0.15
0.15
0.2
0.2
0.2
DharmaFECT
1incells
es at 10,000 cells/well
1. A549
medium
were0containing
plated0 in 96-well
Ham’s
plates
F-12,
2atmM
10,000
L-Glutamine,
cells/well
1.5
in medium
g/L
Sodium
containing
Bicarbonate
Ham’s
and
F-12,
10%
2 mM
FBS,L-Glutamine, 1.5 g/L Sodium Bicarbonate and 10% FBS,
で保存できます。
A
100
100
100
100
100
100
100
100
100
100
100
100
24 hr
before
transfection.
nM siGLO RNA
rmaFECT 1 Transfection
2. siGLO Reagent
Transfection
wereIndicators
mixed in reduced
and DharmaFECT
serum medium
1 Transfection
and incubated
Reagent
forwere
20 min
mixed
at room
in reduced
temperature.
serum medium and incubated for 20 min at room temperature.
C
25
25
25
25
25
25
25
25
25
25
25
25
d to each siGLO/DharmaFECT
3. Complete growth
1 mixture,
medium
and growth
was added
medium
to each
on the
siGLO/DharmaFECT
cells replaced with
1 mixture,
100 µL ofand
resulting
growthtransfection
medium on the cells replaced with 100 µL of resulting transfection
D
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
humidified
incubator,
fixed with
4%
paraformaldehyde,
humidified
washed
with
incubator,
DPBS,
and
fixed
stained
with 4%
withparaformaldehyde, washed with DPBS, and stained with
for 24 hr in 37C 5%
mix.CO
Transfected
cells
were incubated
for
24
hr in100
37C 5%
CO2 100
2 100
E
100
100
100
100
100
100
100
100
100
Hoechst
F
503334250(1 µM).50
50
50
50
50
50
50
50
50
50
G
25
25
25
25
25
25
25
25
25
25
25
25
Transfection Efficiency
C
Transfection Efficiency
C
siGLO Red
Imaging
with
H and
12.5 Analysis
12.5
12.5 Cytell
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
siGLO Red
120
µL/well
MyBioApp by setting
1.
A
the
2-color
Blue
BioApp
Channel
was
to
created
image
nuclei
using
(Hoechst)
MyBioApp
and
by
either
setting
the
the
Orange
Blue
Channel
or
Green
to
Channel
image
nuclei
to
image
(Hoechst)
and
either
the
Orange
or
Green
Channel
to image
120
0.25
0.25
0.25
0.3
0.3
0.3
0.35
0.35
0.35
0.4
0.4
0.4
120
DharmaFECT 1
Figure
1.
96-well
plate
map
of
A549
cell
transfection
conditions.
Triplicate
wells
containing
siGLO
Transfection
Indicator
(12.5,
25,
50,
or
100
nM)
and
siGLODharmaFECT
fluorescence.
1 (0, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, and 0.4 µL/well) are indicated.
100
100
ch overlaps the2.nuclear
Nuclear
area,
reference
was chosen
segmentation,
for the siGLO
which
Transfection
overlaps the
Indicators
nuclear since
area, positive
was chosen
signal
forwill
thebe
siGLO
localized
Transfection
Indicators
since positive signal will be localized
100
As expected, the siGLO Red Transfection Indicatorgelife
Imaging
and
Analysis
with
to the
nuclear
area
ofCytell
eachCell
cell.Imaging System:
siGLO Red
Using MyBioApp a simple 2-color BioApp was developed for each siGLO Transfection Indicator and named siGLO Green and siGLO Red, respectively. Since siGLO
80
results (Figure 4) are very similar tosiGLO
those observed
80
80
Transfection
Indicators
to the nucleus
nuclear
reference segmentation
well of the 96-well
3. Four
plate.
fieldsbecome
werelocalized
captured
fromin positive
eachtransfections,
well of the
96-well
plate. was applied as a mask to sample the siGLO
Pie Chart
Cytell i
fluorescence signal. Example images of negative and positive transfections are shown in Figure 2.
RNA 濃度
one of
e complete, the4.
threshold
After imaging
(linear and
gate)initial
setting
analysis
for positive
were complete,
and negative
the transfection
threshold (linear
was gate)
set immediately
setting for positive
upstream
and negative
transfection
was
set
immediately
upstream
for
siGLO
Green
(Figure
3).
Again,
increased
100nM
Classes 60
100nM
60
60
50nM
histogram chart from
of the
a well
strong
containing
signal observed
a significant
frompositive
a histogram
signal.chart from a well containing a significant positive signal.
transfection is seen by increasing both
siGLO 50nMAll goo
Negative
siGLO Red results:
B
C
%
Transfected
Transfected
%%
Transfected
A
% Transfected
% Transfected
A
Positive
40
40
40
and co
Health
and co
local G
current
25nM
25nM
12.5nM
12.5nM
Transfection
Indicator and DharmaFECT 1. Pie
O and DharmaFECT
Determining
1 amounts
optimal siGLO
for efficient
and DharmaFECT
transfection1ofamounts
A549 cells:
for efficient transfection of
A549 cells:
chart
size
offers
immediate visual indication
ment was set upAintwo-dimensional
a 96-well plate todose-response
determine efficient
experiment
transfection
was set
conditions
up in a 96-well
for A549
plate
cells
to(Figure
determine
1). Aefficient
range oftransfection
increasing conditions for A549 cells
(Figure
1). A
range an
of increasing
20
20
12.5-100 nM) and
amounts
DharmaFECT
of siGLO
1 (0-0.4
Transfection
µL/well)Indicator
were used
(12.5-100
across the
nM)plate
and DharmaFECT
as indicated. A549
1 (0-0.4
cells
µL/well)
were plated
were used
and grown
across for
the plate20 as indicated. A549
were platedwhether
and growneither
for
of cells
determining
reagent had an
armaFECT 1 mixture.
24 hr After
before
anthe
additional
addition24
of hr
thecells
siGLO/DharmaFECT
were fixed, stained
1 mixture.
and analysed
After an
on additional
the Cytell Cell
24 hr
Imaging
cells were
System.
fixed, stained
and analysed on the effect
Cytell Cell
System. Neither high amounts of
The Cyt
Cell Count
onImaging
cell growth.
0
0
100
1 6
2 7
3 8
4 9
5 10
6 11
7 12
82313
0.1
0
493
9
0.15
0.05
0.2
0.25
0.1
0.15
DharmaFECT 1
0.20.3
0.35
0.25
0.3 0.4
0.350.45
DharmaFECT 1 Indicator nor
siGLO
Transfection
トランフェクション試薬
DharmaFECT
1 濃度
DharmaFECT
1 DharmaFECT
1
siGLO Red results:
siGLO Red results:
10
0.4
0.45
DharmaFECT
are for
proced
11
12
Life Tec
1 had significant effect on cell growth under
µL/well
濃
度が
以
上、
および
が 0.15
siGLO
25
nM
DharmaFECT
µl/well
Scienti
4.
Transfection
results
for chart
siGLOview
Red.
A. Pie chart
view showing
negative
positive (red) transfection
Figure
results
for siGLO Red.
A. Pie
negative
(blue) and
positive(blue)
(red)and
transfection
0.1
0.1
0 transfection
0.1 of A5490cells.0.15
0 0.15
0.10.15
0.1
0.1
0.2 was added,
0.15
0.2 4. Transfection
0.15 Figure
0.15
0.2
0.2showing
0.2
Figure
2. Images of negative
A. Negative Controls,
in which no DharmaFECT
1 and up to 100
nM0.2
siGLO Transfection
Indicator
DharmaFECT
1 and positive
these
conditions.
The
heat
maps
for
both
siGLO
As expected,
the siGLO
Red
Transfection
Indicator
are trad
show no nuclear siGLO fluorescence for either siGLO Green or siGLO Red Transfection Indicator; representative image shown. B. Positive transfection staining in the nuclear
In addition,
theset
BioApp
been set upsize
to automatically
size
each
pie diagram
to indicate
results. In addition, results.
the以上のときに、
BioApp
has been
up tohas
automatically
each pie diagram
to indicate
number
of cells number of cells
トランスフェクション効率が
以上になることが
90%
siGLO
Red
region for Hoechst (blue) and siGLO Green Transfection Indicator (green). C. Positive transfection staining in the nuclear
region for both
Hoechst (blue)
and
siGLO
Red
Transfection
expected,
the
siGLO
Red
Transfection
Indicator
counted.
B. Heat
map
showing
percentage
Red
Transfection
Indicator
cells.
C. A graph
of transfect
Dharm
counted.
B.4)
Heat
showing
percentage
of siGLO
Red Transfection
Indicator
positive
cells. C.
Apositive
graph
of transfection
Indicator (orange). 100
100
A
100100
100100
100100 siGLO
100100
100100 As100
100results
100
100
100
100
100
100of siGLO
100
(Figure
aremap
very
similar
to
those
observed
Transfection
Indicators
allow
an
easy
way
to select
Red
分かります。
Pie Chart
efficiency versus DharmaFECT 1 concentration for 4 concentrations of siGLO Red Transfection Indicator.
0
5
nM siGLO RNA
4
0
0
nM siGLO RNA
3
0.05
efficiency
versussimilar
DharmaFECT 1those
concentration
for 4 concentrations of siGLO Red Transfection Indicator.
All othe
results (Figure
4)
are
very
observed
Pie 50
Chart
for siGLO
Green
(Figure 3).to
Again, increased
for evaluating transfectionCompa
Classes50 50
50
50 50
50 50
50
50
50 favourable
50 conditions
50
for
siGLO
Green
(Figure
3).
Again,
increased
Classes Negative
transfection is seen by increasing bothof
siGLO
siRNAs. As can be seen by the large number Electric
March 2
25
25
25
C
25 25
25 25
25 25
25 25 Positive
25 25 Red
25 results:
25
25seen
25 by 25
25
25
25
25
siGLO
transfection
is
increasing
siGLO
Negative
Transfection
Indicator
andboth
DharmaFECT
1.
Piecolored wells, there is a broad range of
of
red
Little Ch
Positive
siGLO
Red
results:
As
expected,
the
siGLO
Red
Transfection
Indicator
Transfection
Indicator
and
DharmaFECT
1.
Pie
12.5
12.5
12.5
D
12.512.5
12.512.5siGLO
12.5Red
12.5
12.512.5
12.512.5
12.512.5
12.5
12.5
12.5 visual
12.5
12.5
12.5
chart12.5
size
offers
an immediate
indication
favourable
conditions.
The
average
percent
of
Cytell
Resource
Center:
siGLO
Red
results:
As expected,
the
siGLO
Transfection
Indicator
GE Healthcare
Cytell Resource Center:
Custom
results
(Figure
4)Red
are
similar
to
those observed
chart
size
offers
an immediate
visual
indication
Pie
Chart
siGLO
Red
ofvery
determining
whether
either
had an100
100
100
100
E
100100 siGLO
100100
100
100
100
100
100100
100
100
100
100 reagent
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直感的に使えるアプリケーションソフトウェア
（BioApps）
Cytell 本体には、画像撮影機能に加え、以下のアプリケーションソフトウェア（BioApps）が標準装備されています。
なお、BioApps は今後さらに拡充される予定です。
解析レポート：解析レポートが自動で作成されます。
専用試薬：専用試薬があります。専用試薬以外でもCytell で撮影可能な蛍光波長に適合する蛍光試薬であればご使用いただけます。
Quick Count
Automated Imaging
オートフォーカス機能があり複数サンプルを同条件で撮影するのに
最適です。
蛍光および透過光撮影ができます。
細胞数計測
蛍光染色が必要です。
ディスポーザブル血球計算板（C-Chip）専用の BioApps です。
Digital Imaging
Cell Viability
蛍光および透過光撮影ができます。
生死細胞解析
蛍光染色が必要です。
Apoptosis
Cell Cycle
1 波長ごとに撮影条件を手動で最適化できます。
NEW
アポトーシス解析
蛍光染色が必要です。
細胞周期解析
蛍光染色が必要です。
GFP
Nuclear Signal
NEW
GFP 陽性細胞率解析
NEW
核内蛍光シグナル解析
蛍光染色が必要です。
核蛍光染色が必要です。
BioApps のワークフロー
数値データ形式
撮影
ウェルごとの結果は、項目ごとにプレートマップ状に表示
解析
細胞ごとのデータも
CSV 形式で保存
レポートアウト
イメージングサイトメーター
Cytell Cell Imaging System
ご注文情報
6,600,000
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Cytell Cell Imaging System（1PC モデル、IN Cell Investigator 付属）
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Cytell Cell Imaging System（2PC モデル、IN Cell Investigator 付属）
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