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14 Agilent Seahorse XFp Glycolytic Rate Assay Kit User Guide
Assay Workflow
Day Prior to Assay
1 For adherent cells, plate the cells at a predetermined
density in the Agilent Seahorse XFp Cell Culture Miniplate
using the appropriate cell culture growth medium. For more
information, refer to the Basic Procedure, “Seeding Cells in
Seahorse XF Cell Culture Microplates”, available on the
Agilent Cell Analysis Learning Center.
www.agilent.com/en/products/cell-analysis/how-to-run-an-
assay
For suspension cells, see “Day of Assay” on page 14.
2 Hydrate a sensor cartridge in Agilent Seahorse XF Calibrant
at 37 °C in a non-CO
2
incubator overnight (refer to Basic
Procedures to Run an XF Assay for details).
Day of Assay
Prepare Agilent Seahorse Glycolytic Rate Assay medium
1
Prepare assay medium by supplementing Seahorse XF
DMEM or RPMI medium with pH 7.4 (Cat No 103575-100 and
103576-100). These media contain the appropriate amount
of HEPES and NO additional HEPES is needed. It is
recommended to start with 1 mM pyruvate,
2 mM glutamine, and 10 mM glucose. However, medium
composition can be changed depending on cell type or the
desired study conditions. For more information, refer to the
Basic Procedure, “Preparing Assay medium for Use in XF
Assays”, on the Agilent Cell Analysis Learning Center.
www.agilent.com/en/products/cell-analysis/how-to-run-an-
assay
2 Bring XF medium with pH 7.4 and XF supplements into a
cell culture hood. Transfer a sufficient volume to a sterile
N O T E
The Cell Line Reference Database is a good resource for finding
information regarding the cell type of interest. Use the link below to obtain
information.
http://www.agilent.com/cell-reference-database
N O T E
If the assay medium is significantly changed from this formulation, the
Buffer Factor Protocol must be performed to derive the buffer factor value.
Consult
Agilent Seahorse XF Buffer Factor Protocol User Guide for more
information.