Agilent Automated Scratch Tool Application Note

Application Note

Cell Migration and
Invasion

Incorporation of a Novel, Automated
Scratch Tool and Kinetic Label-Free
Imaging to Perform Wound

HealingAssays

Author

Brad Larson
Agilent Technologies, Inc.

Abstract

The wound healing or "scratch" assay is one of the most highly used in vitro methods
to monitor and quantify collective cell migration. The current standard involves
manual wound creation, which yields low reproducibility between wounds, high
variability within generated data, and possible false conclusions regarding test
molecules. Using an automated wound creation tool, in addition to kinetic image
capture and analysis, repeatable wounds and robust and repeatable results are
easily attained.

Introduction

Materials and methods

The movement of cells when influenced by interactions
with neighboring cells, otherwise known as collective cell
migration, plays a role in numerous critical physiological
processes, including morphogenesis and tissue regeneration.
This type of movement as a cohesive group has also been
shown to be critical in wound healing and cancer metastasis.
In wound healing, epithelial cells collectively migrate as a
sheet of cells. Wounding of the epithelial layer induces cell
migration in a directional manner. During this process, cells
maintain tight intercellular adhesion, healing the original

2

wound.

Similarly, collective cell migration has also been
implicated as playing a major role in cancer metastasis. An
increasing number of publications indicate that metastatic
cells cluster and invade collectively in the vasculature and

3-4

lymphatics of cancer patients.

Therefore, attaining a better
understanding of collective cell movement is of critical value
for the treatment of multiple disease types.

One of the most widely used methods to measure collective
cell migration is the wound healing or "scratch" assay.
Following creation of a wound, or cell-free zone, within the
confluent cell layer, cell movement back into the wound
area is monitored over time using cellular imaging. Kinetic
and endpoint data then allow for quantification of cell
migration, either when uninhibited or under the influence of
a test molecule. For wound creation, commonly a pipette
tip is manually dragged through the cells, which can lead
to wounds that vary drastically in width, orientation, and in
placement within the well. This yields increased variability in
calculated measurements within replicate wells and across
titrations, complicating final conclusions regarding the
migratory ability of test cell models and treatments, especially
when comparing assay to assay data. To increase the
robustness of generated data, a method to create consistent
wounds is necessary.

This study demonstrates the use of a novel, automated tool
to create scratch wounds in cell monolayers formed on the
bottom of a microplate. With the single push of a button, and
using a 4- or 8-pin head, consistent scratches of equivalent
size and area are made in either 24- or 96-well plates. A
multi-reservoir cleaning trough is also incorporated on the
deck of the tool. Using the onboard programmed procedure,
unattended cleaning and decontamination of each pin is
accomplished before and after use. The small footprint
permits insertion of the tool; using any size laminar flow
hood enabling wound creation in a sterile manner. Following
washing, the plate can then be transferred to an Agilent
BioTek automated imager or the Agilent BioTek BioSpa live
cell analysis system to kinetically monitor cell migration.

Materials

Cells

1

HT-1080 fibrosarcoma cells (partnumberCCL-121)
were purchased from ATCC (Manassas, VA).

Human neonatal dermal fibroblasts expressing RFP

(partnumbercAP-0008RFP) were purchased from
Angio-Proteomie (Boston, MA). U-87 glioblastoma cells

expressing GFP were generously donated by Dr. Sachin Katyal

(University of Manitoba, Winnipeg, Manitoba, Canada).

Experimental components

Advanced DMEM (partnumber12491- 015), fetal bovine
serum (partnumber10437-036), penicillin-streptomycin­glutamine (100x) (partnumber10378-016), TrypLE express
enzyme (1x), phenol red (partnumber12605-010), Alconox
powdered precision cleaner (partnumber16-000-104),
Virkon-S (partnumberNC9821357), and CellTracker Green
CMFDA Dye (partnumberC2925) were purchased from
Thermo Fisher Scientific (Waltham, MA). Cytochalasin
D (partnumber1233) was purchased from Bio-Techne
Corporation (Minneapolis, MN). 24-well clear TC-treated
multiple well plates (partnumber3524) and 96-well

clear, flat bottom, polystyrene TC-treated microplates

(partnumber3598) were purchased from Corning Life
Sciences (Corning, NY).

Agilent BioTek AutoScratch wound making tool

The Agilent BioTek AutoScratch wound making tool
automatically creates reproducible scratch wounds in cell
monolayers grown in microplates. The simple pushbutton
operation and tool-free scratch pin manifold exchange
make it easy to process either 96- or 24-well plates, which
are commonly used in migration and invasion assays. The
compact system features an onboard, preprogrammed
cleaning routine to keep the scratch pins free of buildup and
avoiding contamination. AutoScratch precisely and efficiently
automates the sample prep for imaging workflows with
Agilent BioTek Cytation cell imaging multimode readers and
Agilent BioTek Lionheart automated microscopes.

2

Agilent BioTek Cytation 5 cell imaging multimode reader

Cytation 5 is a modular multimode microplate reader

combined with an automated digital microscope. Filter- and
monochromator-based microplate reading are available, and
the microscopy module provides up to 60x magnification in
fluorescence, brightfield, color brightfield and phase contrast.
The instrument can perform fluorescence imaging in up to
four channels in a single step. With special emphasis on

live cell assays, Cytation 5 features shaking, temperature
control to 65 °C, CO

gas control and dual injectors

2/O2

for kinetic assays and is controlled by integrated Agilent

BioTek Gen5 microplate reader and imager software, which

also automates image capture, analysis and processing.
The instrument was used to capture kinetic high contrast
brightfield and fluorescent images over the incubation period.

Agilent BioTek BioSpa 8 automated incubator

The BioSpa 8 automated incubator links Agilent BioTek
readers or imagers together with Agilent BioTek washers
and dispensers for full workflow automation of up to eight
microplates. Temperature, CO

and humidity levels are

2/O2

controlled and monitored through the Agilent BioTek BioSpa
software to maintain an ideal environment for cell cultures
during all experimental stages. Test plates were incubated in
the BioSpa to maintain proper atmospheric conditions during

incubation and automatically transferred to the Cytation 5 for
high contrast brightfield and fluorescentimaging.

Agilent BioTek MultiFlo FX multimode dispenser

The MultiFlo FX is a modular, upgradable reagent dispenser
that can have as many as two peristaltic pump (8-tube

dispensers), two syringe pump dispensers and a strip washer.

The syringe and washer manifolds can be configured for plate
densities from 6- to 384-well.

Methods

Cell preparation

Cells were cultured in T-75 flasks until reaching 80%

confluency. Subsequent to detachment from the flask
with TrypLE, cells were resuspended to preoptimized
concentrations depending on plate well density and culture

conditions (Table 1).

Table 1. Automated 3D tumoroid invasion imaging
parameters.

Cell Plating Concentrations

24-Well Format 96-Well Format

HT-1080

Fibroblast

U-87

2.4 × 105 cells/mL 4.0 × 105 cells/mL

– 2.0 × 105 cells/mL

2.4 × 105 cells/mL 4.0 × 105 cells/mL

AutoScratch cleaning procedure

Prior to wound creation in test plates, the AutoScratch
tool pins were cleaned and sterilized. The four cleaning
components were added to individual reservoirs of the
cleaning trough, labeled to assist with appropriate component

and volume addition (Table 2).

Table 2. Cleaning trough reagent setup.

AutoScratch Cleaning Components

Reservoir 1

Reservoir 2

Reservoir 3

Reservoir 4

Alconox, 0.5% 12 mL

Virkon-S, 1% 12 mL

Sterile DI H2O 12 mL

70% Ethanol 12 mL

The “Clean” button was pressed to initiate the cleaning
procedure. During the process, the scratching arm containing
the pins moves from the home position into the reservoir

containing 0.5% Alconox, agitates in the Y-axis for 3seconds,
then soaks the pins in the component for 5 minutes. At the
completion of the 5-minute incubation period, the arm moves

the pins to the Virkon-S. The process is then automatically
repeated for each of the remaining components. At the end of
the 20-minute cleaning cycle, the pins were cleaned, sterilized,

and ready to be used for woundcreation.

Scratch wound creation

Following completion of the cleaning procedure, the test plate
was added to the deck of the AutoScratch tool and the lid
removed. The “Scratch” button appropriate for the microplate
density being used, “24” or “96”, was pressed to begin the
wounding process. Here the arm moves the pins from the
home position to column 1 of the plate where a scratch is
made vertically at the center of the well. The arm then moves
the pins back to the reservoir containing the DI H

O and

2

performs a three second agitation to remove any dislodged
cells sticking to the pins. The pins are then moved to

column2 and the scratching and cleaning steps are repeated

for each column of the plate.

3

Post scratch plate washing

Wt = IA – Object Sum Area

t

Upon completion of the wound creation routine, the plate was
transferred to a separate laminar flow hood containing the
Agilent BioTek MultiFlo FX. Here a plate washing procedure
was carried out to remove cells dislodged from the bottom
of the plate. The stainless steel tubes of the strip washer,

previously sterilized using 70% ethanol, were used to aspirate
media while the peristaltic pump and an autoclaved 5 uL

cassette dispensed back fresh media. For uninhibited wells,
the procedure was repeated 3x. For wells containing the
cytochalasin D titration, media containing inhibitor was added
manually following the third aspiration cycle.

Kinetic image-based monitoring of cell migration

Plates were then placed into the BioSpa 8, with atmospheric

conditions previously set to 37 °C/5% CO

. Water was

2

added to the pan to create a humidified environment. The

BioSpa8 software was programmed such that the plates
were automatically transferred to Cytation 5 for high contrast

brightfield or high contrast brightfield and fluorescent imaging
of the test wells, depending on the incorporated cell types.

A single 4x image was taken with each channel (Table 3) to

capture potential cell movement into the original wound area.

Table 3. Included imaging channels per test
cell model.

Incorporated Imaging Channels

HT-1080

Fibroblast

U-87

High contrast brightfield/GFP

High contrast brightfield/RFP

High contrast brightfield/GFP

Plates were then transferred back to the BioSpa 8. Kinetic
imaging cycles were carried out using iterations optimized
depending on the speed of migration for each cell model

(Table 4).

Table 4. Optimized imaging intervals per
cell model.

Kinetic Imaging Intervals

HT-1080

Fibroblast

U-87

Fibroblast/U-87 Co-culture

60 minutes

90 minutes

90 minutes

90 minutes

Table 5. Image preprocessing parameters.

Incorporated Imaging Channels

Channel

High Contrast
Brightfield

RFP

GFP

Apply Image

Processing Background Rolling Ball Priority

Yes Dark 25 μm Fine results

Yes Dark Auto Fine results

Yes Dark Auto Fine results

Cellular analysis of preprocessed images

Cellular analysis was carried out on the processed images
to quantify the cell containing areas of each image using the
criteria in Table 6.

Table 6. Object mask analysis parameters.

Primary Cellular Analysis Parameters

Channel

Threshold

Background

Split Touching Objects

Fill Holes in Masks

Minimum Object Size

Maximum Object Size

Include Primary Edge Objects

Analyze Entire Image

Advanced Detection Options

Rolling Ball Diameter

Image Smoothing Strength

Evaluate Background On

Expand the Threshold Mask

Analysis Metric

Metric of Interest

Tsf[Brightfield]

2,000

Dark

Unchecked

Checked

100 μm

10,000 μm

Checked

Checked

40

20

1% of lowest pixels

5 μm

Object sum area

Wound healing metric calculation

The kinetic cell area coverage values (object sum area) were

then used to generate three additional wound healing metrics,
including wound width, wound confluence, and maximum
wound healing rate. Each metric is automatically calculated

by the Agilent BioTek Gen5 wound healing protocol.

Wound width

Wound width, or the average width of the cell-free zone over
time, is calculated using the following formula:

Image processing

Following capture, using the settings in Table 5, high contrast

brightfield images were processed to increase the contrast
in brightfield signal between background and cell containing
areas of the image, while fluorescent images were processed
to remove background signal.

4

I

H

Where Wt is the average wound width (µm) over time, IA is
the total area of the 4x image, Object Sum Area
covered by cells at each time point, and I

is the area

t

is the height of a

H

4ximage.