Agilent Technologies Seahorse XFp, User Manual

Page: 15 / 24

Assay Workflow
Agilent Seahorse XFp Glycolytic Rate Assay Kit User Guide 15
bottle. It is not necessary to warm the medium and
supplements before this step.
3 Add proper volumes of XF supplements to achieve the
desired final concentrations. This is your assay medium.
When recommended supplement concentrations are used,
pH-adjustment is not necessary.
4
Warm the assay medium to 37 °C in a water bath. It is ready
to use.
Prepare Agilent Seahorse XFp cell culture miniplate for assay
For adherent cells
1
Remove the cell culture miniplate from the 37 °C CO
2
incubator, and examine the cells under a microscope to
confirm consistent plating and proper cell morphology.
2
Wash the cells (refer to Basic Procedures to Run an XF
Assay for details). Remove the cell culture growth medium
in the cell culture miniplate. Wash once with warmed assay
medium using a multichannel pipette, and incubate with
assay medium at 37 °C in a non-CO
2
incubator for 45-60
minutes prior to the assay.
3 Before starting the XF assay, remove the assay medium, and
add fresh, warm assay medium (see
Table 4 on page 17 for
the appropriate Starting Well Volume).
For suspension cells
1
Pellet cells out of their growth medium, and resuspend them
in warm assay medium.
2 Count the cells, and suspend at a concentration such that
seeding 50 µL of cells contains the desired cell number per
well, leaving two wells without cells as background
correction wells.
3
Add 50 µL cells/well, then centrifuge gently to adhere.
4
Gently add 130 µL/well assay medium to each well to match
the Starting Well Volume.
5 Incubate the plate at 37 °C in a non-CO
2
incubator for
45-60 minutes prior to the assay.