Application Note

Enabling Automated Serial Dilutions
Using the Agilent Bravo BenchCel
WorkstationAuthors
Manuel A. Gomez Jr. andChad M. Whitman
Agilent Technologies, Inc.
Santa Clara, CA, USA

Introduction
Many research laboratory protocols require serial dilution of reagents or compounds
for high‑throughput screening experiments. For example, IC50 assays are commonlyused to evaluate enzyme activity as part of the development procedures. Theseexperiments require generating numerous standard curves that involve diluting
proteins, small molecules, as well as other detection agents in large batches.
The required experimental setup requires highly accurate and precise pipettingthat can be streamlined using automated liquid handling equipment with serial
dilution capabilities.
The Agilent Bravo Automated Liquid Handling Platform is ideally suited to handle
an automated serial dilution workflow. It has nine standard deck positions that canhold any ANSI‑standard microplate, and be customized with a variety of accessories
to enable functionality such as heating, cooling, shaking, barcode reading, and tipwashing. The Agilent Bravo Pipetting Head can accommodate volume transfers bycolumn or row, from 300 nL to 200 µL. Further integration of the Agilent BenchCel
Microplate Handler provides an efficient way to store and dispense source anddestination microplates to increase throughput and facilitate more walk‑awaytime. This Application Note describes how these two instruments, acting as anintegrated Agilent Bravo BenchCel Workstation, can provide a complete solution forautomating the creation of serial dilution microplates in column and row formats.
Instrumentation is driven through a preconfigured Agilent VWorks Automation
Control Software protocol that is easy to use and fully enabled for this application.

ExperimentalReagents•Tartrazine (Acros Organics)•Deionized (DI) water

Sample preparation
0.096 grams of Tartrazine was dissolvedin 500 mL of DI water to create a0.36 mM Tartrazine Solution standard.

Agilent VWorks Automation Control
SoftwareTo simplify startup for new users,
preloaded protocols for automated serial
dilution are provided with each Bravo
BenchCel Workstation.

Equipment
The serial dilution workflow was carriedout using an Agilent Bravo BenchCelWorkstation System (Figures 1 and 2)
consisting of:•Agilent BenchCel 4R Microplate
Handler with 660‑mm Front
Loading Racks•

Agilent Bravo Automation Liquid
Handling Platform with Gripper•

Agilent 96LT Disposable PipetteTip Head•

Agilent Bravo Deck Position
Trash Chute•

Agilent 96LT 250 μL Disposable
Pipette Tips•

Agilent 96 Well Manual Fill Reservoir•

96‑well polystyrene, clear flat bottom
microplates (Greiner 655101)•

Nunc 96‑deep well polypropylene,
U bottom microplates(Thermo Fisher 260251)•Thermo Scientific Varioskan Flash2

Figure 1. Agilent Bravo BenchCel Workstation System for Automated Serial Dilution.

Figure 2. Agilent Bravo deck layout for automated serial dilution by column.

Serial Dilution Protocol(column-based)

5.

Unload the 250 μL pipette tips backto Bravo deck position 1.

6.

Press eight 96LT 250 μL disposable
pipette tips onto the last column ofthe head from Bravo deck position 2.

7.

Mix Sample Plate before transferring
into Serial Dilution Plate. Parameters
are three mix cycles of 150 µL, 6 mmfrom the bottom of the plate with a2 µL air gap.

8.

Transfer 150 µL of Tartrazine
solution from Sample Plate intoColumn 1 of the Serial Dilution Plate.
Aspirate 2 mm from the bottom ofthe reservoir with a 2 µL pre‑aspirate
volume. Dispense 2 mm from thebottom of the plate, with a 2 µL
blowout volume. Perform three
mix cycles of 150 µL, 6 mm fromthe bottom of the plate with a 2 µL
air gap.

Instrument setup
Prepare a 96 deep well microplate with
1 mL of tartrazine standard solution
into each well (Sample Plate). Load into
BenchCel Stacker 2.
1.

Open VWorks Software and load theSerial Dilution Protocol.

2.

Load 60 mL of DI water into 96 Well
Manual Fill Reservoir (Bravo deck
position 9).

3.

Load an empty 96‑well polystyrene
microplate (Serial Dilution plate) on
BenchCel Stacker 1.

4.

Load 250 μL pipette tip boxes atBravo deck positions 1 and 2.

5.

Set up a liquid class for a 51–200 μL
dispense in Agilent VWorks
Automation Control Software.

Instrument workflow
1.

Transfer Serial Dilution Plate from
BenchCel Stacker 1 to Bravo deck
position 5.

2.

Transfer Sample Plate from
BenchCel Stacker 2 to Bravo deck
position 4.

3.

Press 250 μL disposable pipette tips
onto the 96LT Head at Bravo deckposition 1.

4.

Transfer DI water from 96 Well
Manual Fill Reservoir into Serial
Diluton Plate in Columns 2–12.
Aspirate parameters are 6 mm fromthe bottom of the reservoir with a2 µL pre‑aspirate volume. Dispenseparameters are 2 mm from thebottom of the plate, with a 2 µL
blowout volume.

9.

Data processing
Absorbance measurements fromeach well were used to determine theprecision of the transfer. Coefficient ofvariance (CV) calculations were madeby dividing the standard deviationby the mean for each column/row.
Accuracy ratio was calculated byaveraging the absorbance reading of two
adjacent column/rows where a pipettedilution occurred.
Results may vary depending on individual
experimental methods and liquidclass optimization.

Perform a 1:2 serial dilution (150 µL)
from Columns 1–10. Aspirate a 2 µL
pre‑aspirate volume 2 mm from thebottom of the plate. Dispense 2 mmfrom the bottom of the plate, with a2 µL blowout volume.

10. Discard 150 µL of excess volume
from Column 10 along with thepipette tips on the Bravo Deck
Position Trash (Bravo deckposition 6).
11. Transfer Sample Plate back to
BenchCel Stacker 4.
12. Transfer Serial Dilution Plate from
Bravo deck position 5 to BenchCel
Stacker 3.
13. Remove Serial Dilution Plate from
BenchCel Stacker 3 and centrifuge at
1,800 rpm for 60 seconds to ensure
consistent well menisci.
14. Perform an absorbance reading
using the Thermo Scientific
Varioskan Flash instrument.

3

Results and discussion
This proof‑of‑principle experiment wasdesigned to demonstrate that the Agilent
Bravo BenchCel Workstation can provide
an automated solution for handlingserial dilution workflows with batchesof microplates. In this experiment, each
Serial Dilution Plate has an estimatedprocessing time of 10 minutes. This
includes the time in which the Serial
Dilution Plates are unloaded from theAgilent BenchCel Microplate Handler
onto the Agilent Bravo Liquid Handlingdeck for the serial dilution steps,
after which they are returned to theappropriate BenchCel stacker.
To demonstrate the precision andaccuracy of the Bravo Liquid Handling
Platform, the CV and accuracy ratio
was calculated for three independent
Serial Dilution Plates (Table 1). Overall,
the average precision and accuracy
ratio was demonstrated to be within
5.0 % of the expected measurement
across the entire plate. A perfect serial
dilution would have an accuracy ratioof 1:2.00 across the entire plate. The
accuracy ratio of the plate improves
with optimal pipetting parameters. For
the column serial dilution experiment,Columns 9 and 10 were removed
from data analysis as the absorbancereadings on these two columns werevery close to the background reading inColumns 11 and 12.

www.agilent.com/chem
For Research Use Only. Not for use in diagnostic procedures.
DE.5949074074
This information is subject to change without notice.
© Agilent Technologies, Inc. 2017, 2020
Printed in the USA, October 29, 2020
5991-8090EN

Table 1. Precision, accuracy ratio, and serial dilution time for three Serial Dilution Plates preparedby column.
CV/Column

Plate
no.

1

2

3

4

5

6

78Average
Time
Accuracy
precision (sec/plate)
ratio

1

1.3 %

1.0 %

2.3 %

1.4 %

2.9 %

2.8 %

3.4 %

6.9 %

2.7 %

568

1:1.95

2

1.4 %

1.7 %

1.8 %

1.6 %

1.5 %

2.5 %

4.5 %

4.4 %

2.4 %

579

1:1.9530.6 %

1.4 %

1.8 %

3.3 %

2.2 %

3.1 %5.2 %

8.6 %

3.3 %

700

1:1.95

Table 2. Precision, accuracy ratio, and serial dilution time for three Serial Dilution Plates
prepared by row.
CV/Row

Plateno.

1

234

56

1

0.7 %

1.8 %

3.0 %

3.4 %

2.4 %

3.0 %

2.4%

517

1:1.89

2

0.5 %

1.6 %

2.6 %

2.5 %

2.8 %

4.7 %

2.5%

455

1:1.8930.7 %

1.8 %

2.5 %

2.2 %

3.5 %

3.3 %

2.3%

453

1:1.89Since the Bravo Liquid Handling Platform
can also perform serial dilutions byrows, a separate experiment was
performed to confirm the consistency inthis orientation. Table 2 shows that by
applying the same pipetting parameters,
high precision and accuracy waspossible with preparing serial dilutions
by rows.
The use of optimal pipetting parameters
for mixing steps before or after a dilution
step contributes to a low CV and a tightaccuracy ratio. The following parameters
are key to obtaining precise and accurate
mixing when using a liquid handling
platform for creating serial dilutions:•Maximize mix volumes•

Maximize mixing speed•

Maximize the area traversed by the
tip retraction/extension

AverageTime
Accuracy
precision (sec/plate)ratioConclusion
The Agilent Bravo BenchCel Workstation
demonstrates a quick and reliable
solution for processing serial dilutions
into multiple microplates across columns
and rows. These experiments showthat optimized pipetting parameters
can display accuracy and precision
when preparing serial dilution samples.
Additionally, the workflow was performed
with a short process time without
any pipette tip changes, minimal
number of mix cycles, and no required
intermediary wash steps. The
automation protocol created in theAgilent VWorks Automation Controls
Software for this experiment provides
a simple template for new users thatcan easily be customized. Overall, theBravo BenchCel Workstation provides
a complete solution for automating
serial dilutions for microplates inhigh‑throughput research environments.