Breakthrough Cell Proliferation Detection: Click Chemistry
Based Labeling of Nucleic Acid Analog
Scott T. Clarke1, Jolene A. Bradford1, Suzanne B. Buck1, Kyle R. Gee1, Brian Agnew1, and Adrian Salic2
1Molecular
Probes® - Invitrogen Detection Technology • 29851 Willow Creek Rd • Eugene, Oregon 97402 • USA 2Harvard Medical School • Boston MA • USA
Introduction
Figure 5 – Colchicine blocked Jurkat cells compared to untreated cells
Changes in cell proliferation as measured by the incorporation of nucleoside analog into actively synthesizing DNA have been the basis for assessing
treatments which alter or block phases of the cell cycle. Initially radioactive thymidine was used and later replaced by bromo-deoxyuridine (BrdU)
incorporation. We introduce a new method for a direct measurement of the S-phase fraction through the use of click chemistry. Here, a labeling strategy
using the Click-iT™ EdU cell proliferation assay from Molecular Probes®, demonstrates that cell proliferation and cell cycle blockers can be assessed by flow
cytometry.
Click chemistry, originally independently defined by Sharpless and Meldal1,2, has been adapted to measuring cell proliferation through the direct detection of
nucleotide incorporation. The assay uses the incorporation of an alkyne modified nucleoside supplied in the growth medium for a defined time period.
Cells are then fixed, permeabilized, and reacted with a dye labeled azide, catalyzed by copper(I). A covalent bond is formed between the dye and the
incorporated nucleotide, and the fluorescent signal can then be measured by flow cytometry, imaging, or high content screening (HCS).
EdU (5-ethynyl-2’-deoxyuridine) based S-phase measurement offers distinct advantages over antibody-based BrdU method. Compared to BrdU, the click
chemistry approach requires less time and sample preparation, and provides greater reliability with at least equivalent sensitivity.
Figure 4 – Cell cycle analysis:
measured vs. modeled data
3A
4A
Cell cycle stain
Figure 3 – Cell proliferation
measured by click labeling
5-ethynyl-2’deoxyuridine
2
Cu (I)
= Alexa Fluor® dye
1
Figure 1 – Click chemistry is the copper(I) catalyzed
reaction between a terminal alkyne and an azide.
A solution containing the dye labeled azide plus
copper(II) and a reductant is added to prepared cells.
Because reactants are abiotic and bio-orthogonal they
are highly selective and inert until catalyzed by
copper. The covalent reaction occurs rapidly at room
temperature.
Click-iT™ cell cycle stain for 633/635 nm excitation fluorescence
Figure 2 – Click-iT™ Work Flow
Figure 3 – Detection of S-phase. HeLa (human
cervical carcinoma) cells were treated with 10 µM EdU
for one hour, harvested, and tested according to
staining protocol using paraformaldehyde fixative and
saponin-based permeabilization reagents.
Panel 3B Dual-parameter labeling of Click-iT™ Alexa
Fluor® 488 azide dye and Click-iT™ cell cycle stain for
633/635 nm excitation. The defined region contains
the actively proliferating cells (%S-phase)
Analyze sample
Figure 2 – The work protocol for click labeling is
simple and similar to that used for standard
immunohistochemistry labeling. Additional steps can
be added for surface and intracellular labeling.
4B
Number
100
3B
0
50
100
150
200
250
(Red for
C-A-SytoxRed
Cycle
Red C-A) fluorescence
Click-iT™ cellChannels
cycle stain
633/635 nm
excitation
Click-iT™ cell cycle stain for 633/635 nm excitation fluorescence
+ colchicine and EdU
Figure 6 – EdU incorporated
cells labeled with click reagent
3
2
5
Figure 6 – Imaging HeLa cells grown in 10 µM EdU for
2 hrs and labeled with Click-iT™ EdU Alexa Fluor® 488
azide (green), α-tubulin/GAM Alexa Fluor® 555
conjugate (red), and nuclear stained with DAPI (blue).
Insert shows control cells grown without EdU and
labeled identically. Arrows indicate proliferating cells.
4
Results and Conclusions
C
A novel method of measuring cell
proliferation by nucleoside incorporation is
presented using click chemistry based
cycloaddition reaction between an alkyne
and an azide.
1
4
4
The thymidine analog EdU provides a
terminal alkyne and reacts with an azide
labeled with a fluorescent compound to form
a covalent bond, thus labeling replicating
DNA
5
D
A simplified work flow enables
Accurate, consistent performance—no
denaturation
steps,
no
harsh
treatments
4
Shorter time to answer—assay
complete in two hours
Dual parameter plot combines staining of DNA
content (x-axis) with EdU label of proliferating cells (yaxis) (panel 3B). Co-positive staining of cells provides
a direct measurement of the percentage of cells
actively synthesizing DNA during the labeling period.
34% of the cells are seen to be actively proliferating.
Figure 5 – Dual-parameter labeling of Jurkat (human T-cell leukemia) cells with Click-iT™Alexa Fluor® 488 azide
dye and Click-iT™ cell cycle stain for 633/635 nm excitation treated with colchicine and EdU. Jurkat cells were
treated ± 1 µM colchicine for 16 hours followed by 10 µM EdU for two hours. Cells were harvested and tested
according to staining protocol using paraformaldehyde fixative and saponin-based permeabilization reagents.
Comparison of control cells without treatments (column 1), cells with colchicine treatment only (column 2), cells
with EdU incorporation only (column 3), and cells with both colchicine treatment and EdU incorporation (column
4). Samples analyzed on a BD LSRII flow cytometer using 488, 405 and 633 nm excitation.
EdU labeling with Alexa Fluor® 488 azide dye shows clear separation between positive label
negative labeled cells 3 (non-proliferating).
2
(proliferating) and
Cells which have been treated with a G2M cycle block (colchicine) show an accumulation of cells in the G2M phase of
the cell cycle 4 with a reduced percentage of cells in the G0G1 and S-phases 5 compared to control cells. This is
seen on the cell cycle plots (row B) as well as the dual parameter plots (rows C and D)
Using the LIVE/DEAD® Fixable Violet Dead Cell Stain to label dead cells before fixation allows gating out of dead
cells, giving a more accurate analysis. This data shows that the cell death was caused by the colchicine treatment
and not by the EdU-incorporation
is
Comprehensive data—new technology
can be multiplexed with antibody based
detection.
Click-iT™ cell cycle stain for 633/635 nm excitation fluorescence
Cell cycle arrest drugs can be screened using
this simple and rapid protocol, compatible
with flow cytometry, imaging, and high
content screening platforms
LIVE/DEAD® Fixable Dead Cell Stains can be
used for gating out dead cell populations.
Row A Histograms show labeling with Click-iT™ Alexa Fluor® 488 azide dye. Row B shows cell cycle labeling using
Click-iT™ cell cycle stain for 633/635 nm excitation. Row C shows combined staining of DNA content with the
labeling of proliferating cells incorporating EdU; co-positive staining of cells 1 provides a direct measurement of the
percentage of cells in the S-phase (DNA synthesis). Dead cells have been gated out in rows A, B, and C by using
the LIVE/DEAD® Fixable Violet dead cell stain. Row D does not have the dead cells gated out (dead cells shown in
orange) and demonstrates how analysis is affected.
G1: 53 %
S: 32 %
G2: 16 %
0
Cell cycle stain
Panel 4B Deconvolution of cell cycle histogram using
ModFit (Verity House) curve fitting software modeling
the percentage of cell population in the G0/G1, S, and
G2/M phases. DNA content histograms require
mathematical analysis such as this in order to extract
the overlapping phase distributions.
300
Perm/wash
Click-iT™ EdU Alexa Fluor® 488 azide fluorescence
Fix/wash
Panel 3A Histogram of cells labeled with Click-iT™
Alexa Fluor® 488 azide dye showing clear separation
of proliferating cells 1 which have incorporated EdU
from non-proliferating cells 2 .
400
Grow cell +EdU
Click/wash
Figure 4 – Normal distribution of cell cycle staining.
Panel 4A Histogram of Jurkat cells stained with ClickiT™ cell cycle stain for 633/635 nm excitation showing
DNA content distribution where G0/G1 and G2/M phase
peaks are separated by the S-phase. Insert shows the
distribution of S-phase (overlay in purple) and G0/G1
and G2/M phases (in green).
Click-iT™ EdU Alexa Fluor® 488 azide fluorescence
200
DNA
+ EdU
B
Click-iT™ EdU Alexa Fluor® 488 azide fluorescence
Figure 1 – Click chemistry based
detection of metabolically
incorporated DNA analog
+ colchicine
Click labeled
A
neg. control
EdU labeling has been demonstrated in both
adherent and suspension cells lines,
including Jurkat, MOLT4, HeLa, COS7,
CHOK1, A549, 3T3, and in phorbitol
stimulated peripheral blood lymphocytes.
References
1.
2.
Rostovtsev, V.V.; Green, L.G.; Fokin, V.V.; Sharpless, K.B.
Angew. Chem. Int. Ed. 2002, 41, 2596
Tornøe, C.W.; Christensen C.; Meldal, M. J. Org. Chem.
2002, 67, 3057