Seeding cells in an XFe24 or XF24 Cell Culture
Microplate
XF assays are performed in Seahorse 24-well XF Cell Culture
Microplates in conjunction with an XF24 sensor cartridge. Each
microplate is formatted in a typical 24-well design, as shown. The
seeding surface of each well is similar to that of a typical 96-well
(0.275 cm2). This procedure describes recommendations for
seeding adherent cell types for use with the XF24 Analyzer.
A two-step seeding process is recommended when seeding XF24 cell culture microplates.
The two-step process produces a consistent and even monolayer of cells.
1. Harvest and re-suspend the cells to desired final concentration to seed in 100 μL of growth
medium. Optimal cell seeding numbers vary widely, though are typically between 10,000 –
80,000 cells per well and must be determined empirically (see Optimization Protocols).
2. Seed 100 μL of cell suspension per well (as shown in figure below); do not seed cells in
background correction wells (A1, B4, C3, D6). Be sure to put medium only (no cells) in the
background correction wells. Allow plate to rest at room temperature in the tissue culture
hood for one hour. This will promote even cell distribution and reduce edge effects.
3. Place plate in a standard cell culture incubator to allow cells to adhere. This generally takes
approximately 1 hour for strongly adherent cells, but may take 5-6 hours for less adherent cell
types. Monitor adherence using a microscope.
4. After cells have adhered, add 150 μl of growth medium to each well (see figure below),
bringing the total volume of medium in the well to 250 μl. When adding medium to the wells,
add it slowly to the sides as not to disturb the newly attached cells.
5. Allow the cells to grow overnight in a cell culture incubator. Monitor growth and health of
cells using a microscope.
Hint: Hold the pipette tip at an
angle and rest the tip just
below the circular rim at the
top of the well for best
technique and most
homogeneous
cell layer.
