Agilent Brave Seahorse Assay Workbench Analyzer User Guide PDF

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Summary of Content for Agilent Brave Seahorse Assay Workbench Analyzer User Guide PDF

User Guide

Introduction Cell-based assays often require several liquid handling steps, which can influence data quality. Lack of consistent sample preparation can result in unacceptable variability across wells, plates, or replicate assays. Such pipetting steps can also be labor-intensive, especially when performing multiplexed or replicate assays in large numbers, as is often required in drug discovery projects.

To address these issues when performing Agilent Seahorse XFe96 assays, several key manual pipetting steps can be automated using the Agilent Bravo automated liquid handling platform with the Bravo Seahorse Assay Workbench software. Performing liquid handling steps using the Bravo platform provides value for the researcher with respect to data quality and reduction in assay preparation (handson) time. Both benefits allow for increased productivity due to more consistent data as well as increased walkaway time during the XF assay.

Seahorse XF assays have common sample preparation elements regardless of the specific application or kit being used:

1. Washing cells (exchanging growth media for assay media)

2. Compound Preparation (dilutions)

3. Compound Port Loading into the XFe96 Sensor Cartridge

These workflow steps are routine, involve substantial pipetting, and have the potential to add error to assays; as such, they are good candidates for automation.

Bravo Seahorse Assay Workbench User Guide

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Required equipment and application software Agilent Seahorse XFe96 analyzer

Agilent Bravo automated liquid handler configured for use with Agilent Seahorse XFe96 consumables (through the Bravo Seahorse assay workbench)

General guidelines The protocols described in this User Guide are designed to work with the Agilent Seahorse XF Bravo deck configuration. An installer for the Bravo Seahorse Assay Workbench software can be acquired by contacting a Bravo Application Engineer. Contact the Automation Technical Support Team at service.automation@agilent.com or 18009794811.

Ensure that the labware is placed on the deck exactly as shown on the form. Failure to place labware in the appropriate deck positions could result in a crash, potentially resulting in damage to the Bravo liquid handling head. Labware needs to be properly seated on each deck position, and the MTC controller should be turned on for temperature control.

Required labware and consumables

Agilent Labware and XF Consumables Part Number

Quantity Required per XFe96 Assay

Cell Washing Compound Dilution Cartridge Loading

24-Column Reservoir (Agilent reservoir, 24 column, polypropylene, 3.25 mL/column, pyramid base geometries)

201296-100 1

96-Well Storage/Reaction Plate (Agilent storage/reaction microplate, 96-well, ultrahigh purity polypropylene, 1 mL/square well, conical bottoms)

201276-100 2

Reservoir (Agilent reservoir, single cavity, polypropylene, 86 mL, 96 pyramids base geometry)

201254-100 2 1

96 V11 LT250 Tip Box (full) 19477-002 1 2 1

96 V11 LT250 Tip Box (empty) 1 1

Agilent Seahorse XFe96 FluxPaka 102416-100 1 cell plate (96 Seahorse

XF96 cell plate) 1 XFe96 cartridge with utility plate (96 Seahorse

XF96 cartridge with utility plate, 384 Ports)

Agilent Seahorse XF assay mediab Assay dependent 90 mL 40 mL 20 mL

Seahorse XF glucose (1.0 M solution)b 103577-100 XF assay dependent

Seahorse XF pyruvate (100 mM solution)b 103578-100 XF assay dependent

Seahorse XF L-glutamine (200 mM solution)b 103579-100 XF assay dependent

Seahorse XF assay kit Application dependent

Table 1. Agilent Bravo labware and XF consumables required for cell washing, compound dilution, and XFe96 cartridge port loading.

a A FluxPak includes 18 XFe96 sensor cartridges, 18 utility plates, 20 cell culture microplates, and 500 mL of XF calibrant. b Choice of XF media used to prepare XF assay media will be XF kit/assay dependent.

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Seahorse XF assay preparation and setup Figure 1 presents a simplified workflow for XF assay setup.

Prepare the XF assay cell plate, sensor cartridge, XF assay media, and XF kit reagents according to the recommended standard procedures.

See the XF Assay Learning Center for all details pertaining to preparation for XFe96 assay materials, including:

Seeding and growth of cells in XF96 cell culture microplates

Hydration of XFe96 sensor cartridges

Preparation of appropriate XF assay media

Preparation of XF kit sensor cartridge injection solutions

Using the Bravo Seahorse Assay Workbench 1. Open the Bravo Seahorse Assay

Workbench.

2. Select the desired protocol: Cell Plate Wash (media exchange), Serial Compound Dilution and Port A Loading, Simple Cartridge Port Loading AD (1 to 4 ports), or Simple Cartridge Port Loading BD (1 to 3 ports), see Figure 2A.

Figure 2. A) Bravo Seahorse Assay Workbench main menu; B) Bravo deck positions.

A B

Figure 1. Agilent Seahorse XF assay setup using the Bravo automated liquid handling platform.

Seed Cells Manual or

Bravo

Wash Cells Bravo

5-15 minutes 5-30 minutes 5-20 minutes

Hands-on time savings with Bravo: 20-60 minutes/plate

Run Assay Seahorse XFe96

Analyzer

Image and Incubate Cytation Imager Imaging and

normalization solution

Dilute Compounds Bravo

Load Cartridge Ports Bravo

Analyze Data Wave software

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3. Follow the directions described in the form.

Set up the Bravo deck with the proper consumables on the appropriate deck position (19, Figure 2B), which will depend on the selected procedure.

Ensure that the MTC controller for heaters in deck positions 4 and 6 is turned on.

4. Start the protocol.

The protocol will immediately begin when Start Protocol is selected. Follow any prompts that appear, for example, modifying the tip state or adding assay media to the appropriate deck location. Once all prompts are satisfied, heaters on positions 4 or 6 will warm to 37 C. This can take several minutes if the heaters were off. Once the deck positions reach 37 C, the protocol will be executed.

Modifying the tip state Some protocols require user input to identify the contents of tip boxes (empty or full). Ensure that the tip boxes are placed on the deck according to the layout before starting the protocol. To modify the tip state, start the protocol, and when prompted:

1. PAUSE the protocol from the dialog box

2. Select TIP STATE EDITOR from the menu on the protocol form

3. Select the tip box position to be edited from the Tip State Editor

4. Click on the three dots that appear on the right to open the Tip Position Dialog box

5. Assign tips according to the form and press OK

6. Close the Tip State Editor

7. CONTINUE the protocol from the dialog box

Protocols

1. Cell plate wash (media exchange)

What the protocol will do: Wash a single XF96 cell culture microplate plate. Remove lid; multiple exchanges (2 to 3) of XF assay media replace the original cell growth media; return lid. The system uses gentle aspirate and dispense speeds to minimize potential cell loss. The XF cell culture microplate is maintained at 37 C throughout the protocol, and kept at 37 C until the protocol complete dialog box is acknowledged.

This protocol requires that cells have been seeded in 80 L of growth media, as recommended for XF96 cell culture microplates. The final volume in the well after the wash is 180 L.

Preparation for running the protocol: Gather labware and consumables

listed in Table 1 for Cell Wash.

Prepare XF assay media with supplements according to the XF assay being performed, and warm to 37 C. A minimum of 80 mL is required to wash a cell plate. The reservoir has a capacity of 86 mL.

Running the protocol: 1. Open the Bravo Seahorse Assay

Workbench, and select Cell Plate Wash. Follow the directions on the screen, and arrange the deck according to the layout on the right side of the screen, shown in Figure 3, and described below.

2. Upon completion of cell plate wash protocol, place the XF96 cell plate in a non-CO2 incubator.

To set up the Bravo deck (Figure 3) 1. XF96 cell plate: Place a microplate

containing cells seeded WITH LID on deck position 6.

2. Assay media: Add 80 mL of 37 C XF assay media to an Agilent 96 pyramid base reservoir plate, then place on deck in position 9.

3. Full tip box: Place a full box (96 V11 LT 250 Tip Box) on deck position 7.

4. Empty Agilent 96 pyramid base reservoir plate: Place on deck position 8.

5. Empty Bravo deck positions (no labware/consumables): Positions 1 to 5.

Figure 3. Cell Plate Wash (Media Exchange) screen. Bravo instructions, deck layout, and labware table as seen in the Bravo Seahorse Assay Workbench for cell plate washing.

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2. Simple cartridge port loading A-D (1 to 4 ports)

What the protocol will do: Aspirate reagents from 24 column reservoir and dispense injection solutions into 1, 2, 3, or 4 ports in the XFe96 sensor cartridge, in the order of A, B, C, and D, respectively. Port loading volumes are: A = 20 L, B = 22 L, C = 25 L, and D = 28 L. Each group of ports (A, B, C, or D) will receive the same injection solution in the respective port, and is intended to be used with standard XF assay workflows with an initial well volume of 180 L. The XFe96 cartridge is maintained at 37 C throughout the protocol, and will remain at 37 C until the protocol complete dialog box is acknowledged.

This protocol may be used for the following XF assay kits and applications (Table 2), as well as custom-designed assays using 1 to 4 ports. In this protocol, all wells receive the same compound in each port. This design would be implemented when the intervention or variable is a medium-to-long-term compound exposure, genetic modification, or comparison of different cell types or treatments made before the XF assay.

Please see Section 4 (Port loading BD) if the assay design calls for a comparison of different compounds/acute treatments to be delivered to the cells through port A.

Preparation for running the protocol: Gather labware and

consumables listed in Table 1 for Cartridge Loading.

Prepare assay media with supplements according to the XF assay being performed, and warm to 37 C. A minimum of 20 mL is required to prepare injection compounds.

Prepare injection solutions according to the XF assay being performed. Injection solutions may be first prepared, then transferred into the 24column reservoir or prepared directly in the 24column reservoir. A 3-mL volume of each injection solution is required to ensure sufficient volume for all 96 ports (Figure 4).

Table 2. A List of common XF assay kits that may be automated with the Bravo platform when using the Simple Cartridge Port Loading A-D (1 to 4 ports) protocol.

XF Kit or Application Ports Used Compounds Used

Seahorse XF Cell Mito Stress Test A > B > C Oligo > FCCP > Rtn/AA

Seahorse XF Real-Time ATP Rate Assay A > B Oligo > Rtn/AA

Seahorse XF Glycolytic Rate Assay A > B Rtn/AA > 2-DG

Seahorse XF Cell Energy Phenotype Assay A Oligo + FCCP

Seahorse XF Glycolysis Stress Test A > B > C Glucose, Oligo, 2-DG

Seahorse XF PMP and Isolated Mitochondria Assays

Coupling A > B > C Oligo, FCCP, AA

Electron Flow A > B > C > D CI substrate* > Rtn > Succinate > Ascorbate/TMPD

Figure 4. 24column reservoir injection solution (port compound) layout for 4port loading protocol (A > B > C > D). Add 3.0 mL injection solution per column. Place Port A compound in Column 1, Port B compound in column 3, Port C in column 5, and Port D in column 7.

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Running the protocol 1. Open the Bravo Seahorse

Assay Workbench, and select Simple Cartridge Port Loading A-D (1 to 4 ports).

2. Follow the directions on the left side of the screen, and arrange the deck according to the layout shown in Figure 5, and described below.

3. Upon completion of the Bravo protocol, begin the appropriate XF assay, and manually insert the cartridge into the XF instrument for calibration when prompted.

To set up the Bravo Deck (Figure 5) XFe96 sensor cartridge: Place a

cartridge with its utility plate and a lid on deck position 4.

Injection solutions: Place the 24column reservoir containing injection solutions in position 5.

Full tip box: Place a full box (96 V11 LT 250 Tip Box) on deck position 2.

Empty tip box: Place an empty tip box (96 V11 LT 250 Tip Box) on deck position 1.

3. Serial compound dilution and port A loading

What the protocol will do A specialized protocol for generating eight individual two-fold serial dilution series. Stock concentrations of each compound are manually loaded into a storage plate in column 1. Each library compound to be diluted is preloaded as a stock into a separate storage plate (the source plate). The Bravo then executes a 10-step two-fold serial dilution of all eight compounds, including distribution of diluent, followed by loading port A of the cartridge with 20 L of all dilutions. Figure 6 illustrates the resulting XF assay plate layout.

Figure 6. Resulting XF assay layout when using the serial compound dilution and port A loading protocol. See Note next page.

Compound concentration

1:2 Serial dilution

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

Compound 8

Intermediate dilution

Vehicle control/ assay media

1:1 103 port 1:1 104 well

Figure 5. Simple Cartridge Port Loading AD screen. Bravo instructions, deck layout, and labware table as seen in the Bravo Seahorse Assay Workbench for loading 1, 2, 3, or 4 injection ports.

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The top concentration in the dilution series, located in column 2, is a 1:1,000 dilution of the stock solution. This translates to 1:10,000 post injection. For example, a 10 mM DMSO stock will generate a dilution series from 10 to 0.02 M (1 to 0.002 M post injection). The XF sensor cartridge is maintained at 37 C throughout the protocol, and will remain at 37 C until the protocol complete dialog box is acknowledged.

Note: By default, solutions from columns 1 (intermediate dilutions of library compounds) and 12 (XF assay media/vehicle control) of the compound dilution plate (deck position 5) will be loaded into the A ports of columns 1 and 12 of the XFe96 cartridge, respectively. If these columns are required for other uses, tips in columns 1 and 12 from the tip box on deck position 8 may be removed before running the protocol, and port A loaded independently for those wells.

This protocol may be followed with the Simple Cartridge Port Loading B-D (1 to 3 ports) to load additional assay injection solutions (for example, XF Cell Mito Stress Test Compounds), described in Section 4.

Preparation for running the protocol 1. Gather the labware and

consumables listed in Table 1 for Cartridge Loading.

2. Prepare assay media with supplements according to the assay being performed, and warm to 37 C.

3. Transfer aliquots of stock test compounds to wells A1 through H1 in column 1 of a 96well storage/reaction plate. A minimum volume of 5 L is recommended for each compound.

Running the protocol 1. Open the Agilent Seahorse Assay

Workbench, and select Serial Dilution and Port A Loading.

2. Follow the directions on the left side of the screen, and arrange the deck according to the layout shown in Figure 7, and described below.

3. Upon completion of the Bravo protocol, begin the appropriate XF assay, and manually load the cartridge into the XF instrument for calibration when prompted.

To set up the Bravo deck (Figure 7) XFe96 sensor cartridge: Place

a cartridge with a lid on deck position 4.

Dilution plate: Place an empty storage plate (Agilent 96well, polypropylene, 1 mL/square well, conical bottom) on deck position 5.

Library source plate: Place the source plate, a storage plate (Agilent, 96well, polypropylene, 1 mL/square well, conical bottoms) containing at least 5 L of each stock library compound (C1 to C8 in plate column 1, Figure 8) onto deck position 6.

Figure 8. Setup for Compound Library Source plate. Place 5 L of each compound to be diluted a well in column 1 of a storage plate.

Figure 7. Serial Compound Dilution and Port A Loading screen. Bravo instructions, deck layout, and labware table as seen in the Bravo Seahorse Assay Workbench for serial dilution of compounds and loading port A with up to eight individual compound dilution series.

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Reservoir for new media: Place a reservoir (Agilent reservoir, single cavity, polypropylene, 86 mL, 96 pyramids base geometry) with 86 mL of appropriate XF assay media (dependent on XF assay performed) on deck position 7.

Full tip boxes: Place full boxes (96 V11 LT 250 Tip Box) on deck positions 2 and 8.

Empty tip box: Place an empty box (96 V11 LT 250 Tip Box) on deck position 1.

4. Simple Cartridge Port Loading B-D (1 to 3 ports)

What the protocol will do Load injection solutions into up to three ports in the XF sensor cartridge in the order: B, C, D*. Each port will receive the same solution in a fixed volume intended to be used with a standard XF assay workflow that assumes an initial well volume of 180 L. Port volumes are B = 22 L, C = 25 L, and D = 28 L. The XFe96 cartridge is maintained at 37 C throughout the protocol, and will remain at 37 C until the protocol complete dialog box is acknowledged.

Note: This protocol assumes that port A has been loaded independently with 20 L of an appropriate injection solution (assay dependent), and is most often used:

After preparing a serial dilution series of several test compounds (see protocol Serial Compound Dilution and Port A Loading).

After loading port A with different compounds/cell treatments (for example, compound library).

This protocol may be used for the following XF assay kits and applications (Table 3), as well as custom-designed assays.

Preparation for running the protocol Gather labware and

consumables listed in Table 1 for Cartridge Loading.

Prepare assay media with supplements according to the XF assay being performed, and warm to 37 C. A minimum of 20 mL is required to prepare injection compounds.

Prepare injection solutions according to the assay being performed. Injection solutions may be prepared, then transferred into the 24column reservoir or prepared directly in the reservoir. Three milliliters of each injection solution is required to ensure sufficient volume for all 96 ports (Figure 9).

* Note: This protocol does not load port A. It is essential that compounds are transferred to/prepared in the specified location in the reservoir (Figure 9) to ensure that the correct compound is loaded in the corresponding port.

Table 3. A list of XF assay kits and applications that may be automated with the Bravo platform when using the Simple Cartridge Port Loading B-D (3 to 4 ports) protocol.

XF Kit or Application* Ports Used* Compounds Used*

Seahorse XF Cell Mito Stress Test (Acute Injection) B > C > D Oligo > FCCP > Rtn/AA

Seahorse XF Real-Time ATP Rate Assay (Induced) B > C Oligo > Rtn/AA

Seahorse XF Glycolytic Rate Assay (Induced) B > C Rtn/AA > 2-DG

Seahorse XF Glycolysis Stress Test (Acute Injection) B > C > D Glucose > Oligo > 2-DG

Seahorse XF PMP and Isolated Mitochondria Assays

Coupling B > C > D Oligo, FCCP, AA

*This protocol assumes port A has been loaded independently with 20 L of an appropriate injection solution (assay dependent).

Figure 9. 24Column reservoir injection solution (port compound) layout for a threeport loading protocol. Place 3 mL of Port B compound in column 3, Port C in column 5, and Port D in column 7.

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Running the protocol 1. Open the Agilent Seahorse Assay

Workbench, and select Simple Cartridge Port Loading B-D (1 to 3 ports).

2. Follow the directions on the left side of the screen, and arrange the deck according to the layout shown in Figure 10, and described below.

3. Upon completion of the Bravo protocol, begin the appropriate XF assay, and manually load the cartridge into the XF instrument for calibration when prompted.

To set up the Bravo deck (Figure 10) XFe96 sensor cartridge: Place the

cartridge with its utility plate and a lid on deck position 4.

Injection solutions: Place the 24column reservoir containing injection solutions in position 5.

Full tip box: Place a full box (96 V11 LT 250 tip box) on deck position 2.

Empty tip box: Place an empty box (96 V11 LT 250 Tip Box) on deck position  1.

Empty deck position (no labware/consumables): Positions 3 and 6 to 9

Figure 10. Simple Cartridge Port Loading BD screen. Bravo instructions, deck layout, and labware table as seen in the Bravo Seahorse Assay Workbench for loading one, two, or three injection ports.

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www.agilent.com/chem/discoverXF

For Research Use Only. Not for use in diagnostic procedures.

This information is subject to change without notice.

Agilent Technologies, Inc. 2019 Printed in the USA, March 20, 2019 5994-0684EN

Resources 1. Maximizing efficiency and

performance of Seahorse XF assays via automation

2. Seahorse XF Assay Learning Center

3. Seeding Adherent Cells in Agilent Seahorse XF96 Cell Culture Microplates

4. Washing Adherent Cells in Agilent Seahorse XF96 Cell Culture Microplates

5. Loading the Agilent Seahorse XFe96

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