Intact colons were dissected from cervically-dislocated mice.

Colons were flushed and placed in pre-oxygenated (95% O₂, 5% CO₂)

Krebs-Henseleit solution at RT

Proximal and distal ends were cannulated to 2 mm

diameter tubes and secured in the center of an organ bath with continuously

oxygenated Krebs-Henseleit solution at 37 ºC.

Syringe pumps were connected to the

inlet and outlet tubes to maintain a flow of solution at a rate of 500 µL/min

through the colon.

The system was allowed to equilibrate for 30 minutes before

recording.

Baseline recordings were taken for 30 minutes, then the Krebs

solution in the organ bath was briefly removed, mixed with C21 to a final

concentration of 2 µM, and replaced in the organ bath. Recordings were taken

for another 30 minutes.