AcuRite 75099, 75111, 75112, 75114 Instruction Manual

Application Note

Cell Analysis

Dynamic Monitoring of Cell Adhesion
and Spreading

xCELLigence real-time cell analysis

Author

Brandon Lamarche,
JoyceVelez, and Leyna Zhao
Agilent Technologies, Inc.

Introduction

The cells that make up the various tissues and organs are held together by specific
molecules that essentially serve as “biological glue”. These molecules confer shape,
structure, rigidity, or plasticity to the cells. During embryogenesis, these biological
molecules, referred to as extracellular matrix (ECM) proteins, serve as “tracks” that
direct cells to the appropriate region within the embryo. This is so they can give rise
to different tissues and organ systems. ECM proteins also play a prominent role
during wound healing and also are involved in directing many important cellular
processes such as proliferation, survival, and differentiation. Failure of cells to
interact with the appropriate biological surface or molecule can be detrimental to the
fate of the cells and can contribute to cancer cell metastases.

The various ECM components, such as fibronectin (FN), collagens (CL),

laminins(LM), and vitronectin (VN), interact specifically with different cells through
specialized cell surface receptors called integrins. Integrins recognize and bind to

specific motifs within the ECM proteins, mediating the ability of cells to specifically
adhere to and interact with the appropriate matrix proteins.1 Integrin receptor
interaction with ECM proteins also begins an intracellular signaling cascade
that directs cellular processes, such as cell survival, proliferation, differentiation,

andmigration.

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The ECM proteins must be purified from
human or animal serum before any
biological effects on cells can be studied.
The purified ECM proteins are then
applied to an appropriate surface, such
as a plastic tissue culture dish or a glass
surface. When applied to an appropriate
surface at low concentrations, the
ECM proteins precipitate and coat the
surface. Cells can be applied to the
coated surface and cellular events,
such as cell adhesion and spreading,
can be assessed by various cellular
and molecular techniques. In general,
these adhesion and spreading assays
determine:

– If a certain cell type can adhere to a

specific adhesive substrate

– Whether the adhesive substrate is

capable of supporting spreading
(a process that requires both
cell adhesion and activation of
intracellular signaling pathways)

– Whether cell adhesion and spreading

are sensitive to specific reagents that
block cell/ECM interaction, interfere
with cell signaling pathways, or
disrupt cytoskeletal architecture

There are several methods for assessing
and quantifying cellular adhesion and
spreading on an ECM-coated surface:

1. The most widely used method
involves applying the cells to
surfaces coated with appropriate
ECM components, allowing the cells
to attach and adhere for a specified
length of time, then washing away the
unbound cells. The attached cells are
then fixed, labeled with fluorescent
reagent, such as rhodamine
phalloidin, and pictured using an
epifluorescence microscope or an
epifluorescence confocal microscope.

2. Alternatively, cells can be labeled
with a dye, such as crystal violet,
and quantified. Quantification
involves either manually counting
the cells under a light microscope
or measuring the absorbance of the

stain after it is solubilized.

3. Cells can also be prelabeled
with a fluorescent dye, such as
6-carboxyfluorescein diacetate
(CFDA), and then applied to an
appropriate ECM-coated surface. The
unbound cells are washed off and
the bound cells are quantified using a
plate reader.

4. A method that is designed to assess
the role of integrins and other
adhesion proteins. This method
involves coating different surfaces
with antibodies or peptides, which
are specific for the various receptors,
then seeding those surfaces with
cells that express the appropriate
integrin receptors. The interaction of
integrin receptors on the cell surface
with the antibody or peptide-coated
surface allows the cells to adhere and
undergo specific morphological and
biological changes. These changes
can then be assessed using one of
the three methods discussed above.

While the assays described above
have been informative, they all have
limitations. All are endpoint assays,
providing only a “snapshot” of the
adhesion process. Further, the assays
involve labor- and cost-intensive
prelabeling or postlabeling of cells.
Finally, they all involve fixation and

permeabilization, which destroys the cell
before it can be analyzed.

The xCELLigence system allows
label-free, dynamic monitoring of cell
events in real time. It addresses some
of the major limitations of the assays
described in this application note. For
instance, because the technique is
noninvasive, it does not require the cells
to be fixed or lysed. That means it can
be used to monitor biological events that
occur after adhesion and spreading, such
as proliferation and differentiation.

In this application note, a series of
experiments is described to demonstrate
that this new impedance-based system
is suitable for monitoring cell adhesion
and spreading.

Materials and methods

Cells

All the cells used in this study were
obtained from ATCC and maintained in a

37°C incubator with 5% CO2 saturation.

NIH3T3 cells were maintained in DMEM

media containing 10% FBS, 1% penicillin,
and 1% streptomycin. Jurkat T cells and

BxPC3 cells were maintained in RPMI

containing 10% FBS, 1% penicillin, and
1% streptomycin.

Cell adhesion assays using
impedance technology

The indicated concentration of either
FN or the control PLL was added into
the wells of 96-well E-Plates, then the
plates were incubated for one hour at

37°C. The protein‑coated plates were

washed with PBS and incubated with

0.5% BSA solution in PBS for 20 minutes
at 37°C. The wells of the treated plates

were washed with PBS before media and

cells were added. Cells were trypsinized,

spun, resuspended in serum-free media

containing 0.25% BSA and adjusted to
an appropriate concentration. A 100µL

volume of the cell suspension was
transferred to ECM- or PLL-coated wells
on E-Plates. The extent of cell adhesion
and spreading, measured as changes
in impedance with the xCELLigence

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system, was monitored every three

PLL

minutes for 1to3 hours depending

on the experiment. The assay system
expresses impedance in arbitrary Cell
Index (CI) units. The CI at each time
point is defined as (Rn – Rb)/15, where
Rn is the cell-electrode impedance of the
well when it contains cells and Rb is the
background impedance of the well with
the media alone.

Treatment with inhibitors

For each inhibitor, cells were

pre‑incubated for 15to30 minutes with

the indicated inhibitor concentrations
and then added to ECM-coated wells of
E-Plates. All other steps were the same
as previously mentioned.

1.2

A

Cell Index

Poly-L-

1.0

Fibronectin

0.8

0.6

0.4

0.2

0

0 0.5 1.0 1.5 2.0 2.5

Time (hours)

siRNA Transfection

BxPc3 cells were transfected with 20 nM

of siSRC using siPORTamine at a final
volume of 60 µL. Cells were assayed

for adhesion function 48 hours after
transfection.

Immunofluorescence and light
microscopy

Cells were seeded into PLL- or FN-coated
16-well chamber slides. The cells
were allowed to attach, and then were

fixed with 4% paraformaldehyde at

the indicated time points. The cells

were permeabilized, stained with

rhodamine-phalloidin, then photographed
using an epifluorescence microscope
connected to a digital camera.

Results and discussion

Dynamic monitoring of cell adhesion
and spreading on different surfaces
using impedance technology

To assess the extent of adhesion and
spreading, E-Plates were coated with
either FN or PLL (control). NIH3T3 cells
were applied to the coated wells and the
extent of adhesion and spreading was
monitored using the impedance-based
system. Simultaneously, chamber slides
were also coated with FN or PLL and
the same number of cells were added
to each well. To assess cell attachment
and spreading, cells were stained with

rhodamine‑phalloidin and analyzed with

an epifluorescence microscope.

As shown in Figure 1A, the Cell Index (CI)
increased dramatically when cells are
applied to FN-coated wells. In contrast,
the CI increased slowly and steadily
when cells are applied to PLL-coated
wells. Similarly, immunofluorescent
images (Figure 1B) showed that cell
attachment on FN was accompanied by
immediate spreading. The spreading was

maximal after one hour. On PLL‑coated

wells, the cells tend to remain round even
two hours after initial attachment.

B

5 minutes 30 minutes 60 minutes 120 minutes

25 µm 25 µm 25 µm 25 µm

FN

25 µm 25 µm 25 µm 25 µm

Figure 1. (A) Dynamic monitoring of cell attachment and spreading on PLL- and FN-coated surfaces. (B) The Cell Index correlates
with the extent of cell attachment and spreading observed using conventional phalloidin staining of the actin cytoskeleton and
immunofluorescence microscopy.

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