BioProbes 75
TOOLS FOR FLUORESCENCE IMAGING
Cancer biology in the third dimension
Fluorescent reagents for staining 3D cell cultures created with Cell-Mate3D matrix.
Beth Lindborg, Caitlin Johnson, Yi Wen Chai, Eu Han Lee, and Scott Brush (BRTI Life Sciences); Brian Almond (Thermo Fisher Scientific).
Unlike their 2D tissue culture counterparts, tumors and their associated
When Cell-Mate3D Dry Blend is combined with cells resuspended
microenvironments contain a highly complex and dynamic set of inter-
in Cell-Mate3D Hydration Fluid, the matrix forms instantly through elec-
actions between different cell types [1], multiple chemical gradients [2],
trostatic interactions between carboxyl groups on the hyaluronic acid
and a variety of extracellular matrix components [3,4]. In addition to the
and the amine groups on the chitosan. The resulting poly-electrolytic
biochemical reactions that take place, a tumor’s physical character-
complex is a fibrous matrix that exhibits tissue-like stiffness, emulating
istics, including the rigidity or stiffness of the extracellular matrix, can
the natural cell environment. The Cell-Mate3D matrix enables modeling
regulate its physiology [5–7]. The next discoveries in cancer biology
of cell migration and proliferation; laboratory techniques such as cell
and treatment will require tools that help to create more relevant and
staining and imaging, flow cytometry, and scanning electron microscopy
predictive models as well as methods to effectively analyze them.
can be used with this technology [8].
The combination of 3D cell culture techniques with methods
for analyzing cell health promises to deliver new insights into cancer
Determine cell viability in 3D cultures
mechanisms and new strategies for interrupting or subverting critical
The Invitrogen ™ LIVE/DEAD ™ Viability/Cytotoxicity Kit provides a
pathways. Here we describe the application of fluorescence-based
well-established fluorescence assay for determining viability in a wide
assays and protocols to a complex 3D environment, often with only minor
range of animal cells. The LIVE/DEAD viability assay comprises two
adjustments to exposure time or reagent concentration. Fluorescent
fluorescent probes—calcein AM, a cell-permeant esterase substrate,
reagents from Thermo Fisher Scientific can be used to assess cell
and ethidium homodimer-1, a cell-impermeant high-affinity nucleic
function—including viability, proliferation, and apoptosis—in response
acid stain—that are used simultaneously to stain cells. We used the
to various 3D culture conditions and treatments.
LIVE/DEAD viability assay with 3D cell cultures that were created by
embedding AU565 breast cancer cells into Cell-Mate3D matrix. After the
Create 3D cultures with Cell-Mate3D matrix
matrix with embedded cells was incubated for 4 days, one sample was
Cell-Mate3D™ matrix from BRTI Life Sciences is a tissue-like matrix
left untreated and an equivalent sample was treated with 0.5% Triton™
that offers researchers a biologically relevant and chemically defined
X-100 detergent, which disrupts cell membranes and kills cells. Both
microenvironment for in vitro and in vivo biomedical research. The
samples were then assayed using the LIVE/DEAD Viability/Cytotoxicity Kit
Cell-Mate3D matrix is composed of two naturally occurring biopoly-
and the supplied protocol, except that the incubation time was reduced
mers: chitosan, a positively charged polysaccharide derived from
from 30 to 20 minutes to optimize staining levels in this system. After
chitin, found in the exoskeleton of crustaceans; and hyaluronic acid,
counter­staining with a blue-fluorescent nucleic acid dye, the samples
a linear polysaccharide found in the extracellular matrix of connective,
were imaged on an inverted confocal microscope (Figure 1). The untreated
epithelial, and neural tissues. Hyaluronic acid is typically involved in cell
sample shows live cells labeled with green fluorescence, whereas the
proliferation, migration, embryonic development, and wound healing.
treated sample shows dead cells labeled with red fluorescence.
Figure 1. Detection of viable cells in Cell-Mate3D matrix using the LIVE/DEAD
viability assay. AU565 breast cancer cells growing in Cell-Mate3D matrix were
stained using the Invitrogen™ LIVE/DEAD™ Viability/Cytotoxicity Kit (Cat. No. L3224)
and counterstained using Invitrogen™ NucBlue™ Live ReadyProbes™ Reagent (Cat.
No. R37605). (A) In the untreated sample, live cells fluoresce green and nuclei
fluoresce blue; very few nonviable (red-fluorescent) cells are detected. (B) After
treatment with 0.5% Triton™ X-100 detergent, the sample contains primarily nonviable (red-fluorescent) cells; the purple color of the cells in the image represent
the overlap of the red- and blue-fluorescent stains. Samples were imaged using an
inverted confocal microscope at 20x magnification.
© 2017 Thermo Fisher Scientific Inc. All rights reserved. For Research Use Only. Not for use in diagnostic procedures.
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BioProbes 75
Detect cell proliferation in 3D cultures
In addition to its essential role in development, cell proliferation is an important marker of cancer
cells and can serve as a target of anti-cancer therapies. The Invitrogen™ Click-iT™ EdU Imaging
Kits provide a simple, efficient proliferation assay that detects DNA synthesis using fluorescence
microscopy. In the Click-iT EdU assay, a modified thymidine analog (5-ethynyl-2´-deoxyuridine, or
EdU) is introduced into cells, incorporated into newly synthesized DNA, and then labeled with a
brightly fluorescent Invitrogen™ Alexa Fluor™ dye in a fast, highly specific click reaction. We used
the Click-iT EdU Alexa Fluor 488 Imaging Kit (with no changes to the supplied protocol) with 3D
cell cultures that were created by embedding HeLa cells into Cell-Mate3D matrix, and found that
proliferating cells (which fluoresce green) were easily visualized in 4-day-old cultures (Figure 2).
Detect apoptosis in 3D cultures
The Invitrogen™ CellEvent™ Caspase-3/7 Green Detection Reagent enables quick and reliable
quantitation of apoptotic cells in culture. This nonfluorescent substrate is composed of the
4–amino acid peptide DEVD, which contains the caspase-3/7 recognition site, conjugated to a
Figure 2. Detection of proliferating cells in the
Cell-Mate3D matrix using Click-iT EdU staining.
HeLa cells were embedded in the Cell-Mate3D
matrix and cultured for 4 days. Proliferating cells were
detected using the Invitrogen™ Click-iT™ EdU Alexa
Fluor™ 488 Imaging Kit (green, Cat. No. C10337).
Cells were counterstained with the Invitrogen ™
NucBlue™ Fixed Cell ReadyProbes™ Reagent (blue,
Cat. No. R37606), and imaged using an inverted
confocal microscope at 60x magnification.
nucleic acid–binding dye. In the presence of activated caspase-3 or -7, the substrate is cleaved,
freeing the dye to bind to DNA and producing a bright green-fluorescent signal indicative of
apoptosis. To test this reagent with 3D cell cultures, we embedded AU565 breast cancer cells
in CellMate-3D matrix and cultured the cells for 12 days. During this time, one sample was
left untreated and an equivalent sample was treated with 100 µM paclitaxel (generic name of
Taxol™ pharmaceutical) to induce apoptosis. Samples were then stained with 15 µM CellEvent
Caspase-3/7 reagent (3 times the recommended concentration). Green-fluorescent apoptotic
cells were clearly seen in the paclitaxel-treated sample by confocal microscopy (Figure 3).
Apply fluorescence protocols to your 3D experiments
Although some optimization was required when applying fluorescent probes to 3D cell
cultures growing in Cell-Mate3D matrix, we found that generally the reagents were able to
penetrate the complex environment and accurately report cell health parameters and enzyme
activity. Analysis of cell function within 3D cell cultures will greatly benefit from the plethora
of fluorescent cell function probes available. Explore fluorescence-based cell analysis at
thermofisher.com/cellanalysisbp75. ■
Figure 3. Detection of apoptotic cells in the
Cell-Mate3D matrix using CellEvent Caspase-3/7
Green reagent. AU565 breast cancer cells were
embedded in the Cell-Mate3D matrix and cultured for
12 days. During this time, cells were (A) left untreated
or (B) treated with 100 µM paclitaxel to induce apoptosis. Cells were then incubated with the Invitrogen™
CellEvent™ Caspase-3/7 Green Detection Reagent
(15 µM, Cat. No, C10423) for 30 min to label apoptotic
cells with green fluorescence, counterstained with the
Invitrogen™ NucBlue™ Live ReadyProbes™ Reagent
(blue, Cat. No. R37605), and imaged using an inverted
confocal microscope at 20x magnification.
Acknowledgments: This article was contributed by Beth Lindborg, Caitlin Johnson, Yi Wen Chai,
Eu Han Lee, and Scott Brush, BRTI Life Sciences, Two Harbors, Minnesota (brtilifesciences.com).
Images were acquired at the University of Minnesota-University Imaging Center (UMN-UIC) facility;
Product
Quantity
Cat. No.
we greatly appreciate UMN-UIC member Grant Barthel’s assistance with confocal microscopy.
LIVE/DEAD™ Viability/
Cytotoxicity Kit, for
mammalian cells
1 kit
L3224
CellEvent™ Caspase-3/7
Green Detection Reagent
25 µL
100 µL
C10723
C10423
Click-iT™ EdU Alexa Fluor™
488 Imaging Kit
1 kit
C10337
NucBlue™ Live
ReadyProbes™ Reagent
1 kit
R37605
NucBlue™ Fixed Cell
ReadyProbes™ Reagent
1 kit
R37606
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12 | thermofisher.com/bioprobes
© 2017 Thermo Fisher Scientific Inc. All rights reserved. For Research Use Only. Not for use in diagnostic procedures.