Agilent XF Palmitate Oxidation Stress Quick Start Guide

Quick Start Guide

Response to inhibitor at maximal respiration

XF Palmitate Oxidation Stress Test Kit: Advanced Assay

Day 1 Day 3Day 2

Chronic intervention(s)

e.g., genetic manipulation, chronic drug exposure

Cell seeding

Substrate Limitation

Figure 1. Advanced assay design for use with the XF Palmitate Oxidation Stress Test Kit (p/n 103693-100)

Two day prior to the assay (Day 1)

1. For adherent cells, plate cells at a predetermined density in cell culture growth medium.

2. Use the XF Substrate Oxidation Stress Test- Advanced assay template to design experiment in Wave and make any necessary modifications to the template to suit experimental design.

One day prior to the assay (Day 2)

1. Ensure the XF Analyzer is powered on and thermally equilibrated to 37 °C (minimum of 5 hours).

2. Hydrate a sensor cartridge in sterile or distilled water at 37 °C in a non-CO2 incubator overnight.

3. Prepare appropriate volume of Substrate-Limited Growth Media (~15 mL, Table 1).

4. Aspirate growth media from cell plate and replace with Substrate-Limited Growth Media (100 µL for 96-well plates, 250 µL for 24-well plates); incubate overnight.

Day of assay (Day 3)

1. Complete sensor cartridge hydration: replace water with XF calibrant (200 µL/well for XF96 or 500 µL/well for XF24) and incubate at 37°C, no CO2 , for 1 hr.

2. Prepare 75 mL Assay Media: Supplement XF DMEM or XF RPMI with XF Agilent substrates (Table 2).

3. Aspirate media from cell plate and replace with SubstrateLimited Assay Media: 180 µL for 96-well plates, 500 µL for 24-well plates (Note: Palmitate and BSA will be added just prior to the assay, step10).

4. Place cell plate in non-CO2 , 37 °C incubator for 60 min, or place in Biotek instrument for normalization.

Acute intervention(s)

(Pretreatments) e.g., compound exposure

400

Etomoxir or

medium

300

200

OCR (pmol/min)

Basal

100

Lower

substrate

demand

0 20 40 60 80 100

Figure 2. Advanced assay output

Base growth media

DMEM or RPMI without glucose, pyruvate, glutamine, or GlutaMAX

Tab le 1. Suggested initial Substrate-Limited Growth Media composition.*

* Optimal li mited substrate concentrations and optimal time of incubation a re cell

dependent and should be empirically determined for the cell type of interest.

Glucose/pyruvatre Oxidation Stress Test

Basal>ETO>Oligo>FCCP>Rtn/AA Media = DMEM + Palmitate/BSA + Glucose

Oligomycin

BSA BSA + Eto

Palmitate Palmitate + Eto

Non-mitochondrial oxygen consumption

Time (minutes)

Growth media supplement

Glucose 0.5 mM

Glutamine or GlutaMAX Serum (e.g., FBS)

XF L-Carnitine 0.5 mM

FCCP

Accute response

Response to inhibitor at basal respiration

Smaller response range

(∆OCR)

Suggested initial concentration in SubstrateLimited Growth Media

1.0 mM

1.0 %

Rotenone/

antimycin A

Maximal

Higher

substrate

demand

Inhibited maximal

Sub Ox Analytics >

Basal, Acute and Maximal OCR

Maximal response

Larger response

range(∆OCR)

5. Prepare stock solutions: resuspend dry compounds in assay media and vortex for ~1 min (Table 3).

6. Using stock solutions, prepare 10X working solutions by mixing stock solutions with the appropriate amount of assay media (Table 3).

7. Pipette the 10X working solutions into the each of the four injector ports (Table 3). Note: Use assay media in Port A for the

control (i.e., – etomoxir) wells.

8. Open Wave and the designed assay template. Click Start Run when you are ready.

9. When sensor cartridge calibration is complete, aspirate media from cell plate and replace with Substrate-Limited Assay Media: 150 µL for 96-well plates, 415 µL for 24-well plates

10. Add Palmitate-BSA or BSA control to appropriate cell plate wells: 30 µL for XFe96, 85 µL for XFe24.

11. When prompted, place the loaded sensor cartridge into the analyzer and click I’m Ready.

12. Following calibration, Wave will display Load Cell Plate. Click Open Tray, then replace the Utility Plate with the Cell Plate.

13. Ensure the lid is removed from the Cell Plate, then click Load Cell Plate to start the assay.

14. Optional: Perform post-assay cell normalization using the Biotek instrument.

Assay media component Volume Final concentration (mM)

Seahorse XF DMEM or RPMI Medium, pH 7.4

XF Glucose (1 M) 150 µL 2.0

XF L-Carnitine 75 µL 0.5

Tab le 2. Suggested initial Substrate-Limited Assay Media. Note that Palmitate-BSA and BSA control are added just prior to the assay, see Step 10.

75 mL -

Stock solution 10X working solutions for injection ports

Port Compound

A Etomoxir 700 500 1500 20/56 4.0

B Oligomycin 420 300 2700 22/62 1.5

FCCP

(use optimal concentration determined prior to assay)

D Rotenone + antimycin A 540 300 2700 27/75 0.5

Tab le 3. Standard Substrate Oxidation Stress Tests: Stock and Working solutions.

Volume of assay medium (µL)

720

Stock volume (µL)

75 2925 25/69 0.25

150 2850 25/69 0.5

300 2700 25/69 1.0

600 2400 25/69 2

Volume of assay medium (µL)

Volume added to port (µL)

XFe96/XFe24

Ordering Information

Description Part Number

XF Palmitate Oxidation Stress Test Kit 103693-100

Seahorse XF DMEM Medium, pH 7.4 103575-100

Seahorse XF RPMI Medium, pH 7.4 103576-100

Seahorse XF 1.0 M Glucose 103577-100

Additional information

Final well concentration (µM)

XF Substrate Oxidation Stress Test Kits User Manual:

www.agilent.com/chem/subox-usermanual

Agilent XF Learning Center:

www.agilent.com/en/products/cell-analysis/how-to-run-an-assay

Technical assistance:

cellanalysis.support@agilent.com

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

This information is subject to change without notice.