Thermo Fisher Scientific • 29851 Willow Creek Road • Eugene, OR 97402 • thermofisher.com
The microtubule cytoskeleton is of interest to researchers studying
numerous cellular functions with live cell fluorescence microscopy.
Currently, microtubules are visualized in live cells by way of
fluorescent proteins (FPs) fused to tubulin monomers, which are
subsequently transfected and overexpressed thus providing a
number of challenges inherent to this approach. For instance,
transfection can induce cellular stress and cytotoxicity, while
transgene expression from a plasmid or viral delivery requires
significant time before sufficient protein is expressed to generate
signal that is suitable for live cell imaging. Additionally, fluorescent
proteins are largely limited to the green and red channels, thus
preventing the use of these commonly used channels for other
labels of interest. Most notably, overexpression results in vastly
heterogeneous expression levels, yielding uneven labeling across
the sample and making quantitative characterization of the
microtubule cytoskeleton intractable by fluorescence imaging.
To answer these and other needs, here we describe Tubulin
Tracker™ Deep Red; a novel, membrane permeable fluorescent
molecule with deep red emission and specificity for microtubules in
live cells. Tubulin Tracker Deep Red mitigates many of the
challenges associated with FP-based microtubule labeling,
bypassing the need for transfection and thus providing uniform
labeling of microtubule cytoskeleton in live cells with a rapid
workflow. Our data demonstrates that Tubulin Tracker Deep Red
can be used to label microtubules in live cells in as little as 30
minutes, with no measurable cytotoxicity up to 24 hour incubation.
We also demonstrate the ability to use the label in a ‘no wash’
protocol, enabling quantitative analysis of neurite outgrowth in a
primary neuronal culture model as well as assessment of
pharmacological effects of microtubule destabilizing drugs.
Combined with advanced photobleach protection properties, plus
the enhanced depth permeation in 3D spheroid models and low
phototoxicity of deep red emitting labels, Tubulin Tracker Deep Red
is suitable for long term time lapse imaging as well as rapid labeling
of tubulin for endpoint assay analysis.
CONCLUSIONS
Oggie Golub, Jongtae Yang, Schuyler Corry, Bhaskar Mandavilli, Dan Beacham - Thermo Fisher Scientific, Eugene, OR, 97402
Visualizing microtubule dynamics in live cells using a novel, deep red cytoskeletal label
P2847
Figure 1. Tubulin Tracker Deep Red demonstrates
superior photostability in live cells
Figure 3. Tubulin Tracker Deep Red enables quantitative
analysis of 3D neurosphere maturation
Figure 4. Tubulin Tracker Deep Red enables extended
time lapse imaging in various cell types
Figure 5. Tubulin Tracker Deep Red enables neurite
outgrowth pharmacological response assays
Figure 2. Tubulin Tracker Deep Red demonstrates
minimal cytotoxicity and superior retention in live cells
For Research Use Only.
Not for use in diagnostic
procedures.
Tubulin Tracker Deep Red can be used to visualize microtubules in a
wide variety of live cell types, including 3D spheroid models, and its
far-red spectra properties enable it to be multiplexed with many
common fluorophores and fluorescent proteins. When compared to the
existing live cell Tubulin labeling methods, Tubulin Tracker Deep Red
provides uniform staining, superior photobleach resistance, and no
measurable cytotoxic effects even after 24 hour incubation. This
unparalleled performance enables the study of microtubule dynamics by
performing extended time lapse imaging, as well as pharmacological
investigations of microtubule networks.
ABSTRACT RESULTS
t=0 t=30 sec t=60 sec
Tu
b T
rack
er
D.R
ed
Tu
b T
rack
er
Gre
en
Tubulin Tracker Photostability
Time (s)
Avera
ge F
luo
rescen
ce In
ten
sit
y (
RF
U)
0 10 20 30 40 50 600
100
200
300
400
500
600
700
800
900
1000
Tubulin Green
Tubulin D.Red
Tubulin Tracker Viability (HeLa Cells)
Pre
sto
Blu
e™
RF
U (
56
0/5
90)
DM
SO
Pac
litax
el
Doce
taxe
l
Tubulin D
.Red
Tubulin G
reen
DM
SO
Pac
litax
el
Doce
taxe
l
Tubulin D
.Red
Tubulin G
reen
0
50
100
150
2002 hr
24 hr
Tubulin Tracker Viability (HASM Cells)P
resto
Blu
e™
RF
U (
56
0/5
90)
DM
SO
Pac
litax
el
Doce
taxe
l
Tubulin D
.Red
Tubulin G
reen
DM
SO
Pac
litax
el
Doce
taxe
l
Tubulin D
.Red
Tubulin G
reen
0
20
40
60
802 hr
24 hr
DMSO Paclitaxel Docetaxel Tubulin Tracker D.Red Tubulin Tracker Green
HA
SM
@24
hr
+ CultureOne
Neu
rob
asa
l P
lus
Neu
rob
asa
l
Tu
bu
lin
D.R
ed
1:1
0,0
00 –
no
wash
Mitosis Time Lapse Microtubule Dynamics & Cell Proliferation
Tu
bu
lin
D.R
ed
1:1
0,0
00 –
no
wa
sh
Neurite Outgrowth Neuronal Microtubule Dynamics
Live mouse embryonic hippocampal neurons cultured 14 DIV (days in
vitro) were imaged across 72 hours at 5 minute intervals. Left panel
shows neuronal dynamics; right panel shows a zoomed inset of
individual neurons monitoring neuronal outgrowth activity.
HeLa cells and HASM (Human Arterial Smooth Muscle) cells were
incubated with 1 µM unconjugated taxanes and Tubulin Trackers for
24 hours, and cellular viability was assessed using PrestoBlue
reagent on a Varioskan LUX multimode plate reader. Cellular
proliferation was assessed using anti-Ki67 and Click-iT EdU staining
on a CellInsight CX5 high content analysis platform.
HeLa cells were labeled with each Tubulin Tracker as recommended,
and imaged continuously for 60 seconds on the EVOS FL Auto 2
using a 20x/0.75 NA Olympus SApo objective.
Neural stem cell (NSC) derived neurospheres were cultured using
Gibco Neurobasal and Neurobasal Plus with and without CultureOne
Supplement and stained with Tubulin Tracker Deep Red to assess
functional maturation of progenitors into mature neurons.
Tubulin Tracker Proliferation (HeLa Cells)
% P
osit
ive C
ells
DM
SO
Tubulin D
.Red
Tubulin G
reen
DM
SO
Tubulin D
.Red
Tubulin G
reen
0
20
40
60
80
100Ki67
EdU
Tubulin Tracker Proliferation (HASM Cells)
% P
osit
ive C
ells
DM
SO
Tubulin D
.Red
Tubulin G
reen
DM
SO
Tubulin D
.Red
Tubulin G
reen
0
20
40
60
80
100Ki67
EdU
Live HeLa cells labeled with 100 nM Tubulin Tracker Deep Red and
imaged across 72 hours at 20 minute intervals. Left panel shows a
zoomed out view of cellular movement, microtubule dynamics, and
proliferation; right panel shows a zoomed inset of two mitotic cells.
- CultureOne Neurobasal Plus + CultureOne Control 10 µM Cadmium 31.6 µM Cadmium 100 µM Cadmium
Live mouse embryonic hippocampal neurons cultured 14 DIV (days in
vitro) were treated in half-log intervals with Cadmium Chloride for 18
hours and stained using Tubulin Tracker Deep Red to assess neurite
morphology on the CellInsight CX5 High Content Analysis platform.
Cadmium [µM], log10
Mean
Sig
nal In
ten
sit
y (
RF
U)
-2 0 2 4100
225
350
475
600
Tub Tracker D.Red Intensity
Cadmium [µM], log10
-2 0 2 40.50
0.75
1.00
1.25
1.50Branch Point / neuron
Co
un
t (A
bso
lute
)
Cadmium [µM], log10
-2 0 2 40
25
50
75
100 Neurite area
Are
a (
µm
2)
New - Tubulin Tracker Deep Red Existing Tubulin Tracker Green
U2OS (uniform staining) Cortical neurons U2OS
HeLa (interphase & mitosis) U2OS (with GFP & RFP) HASM (primary cells)