D 1841

Gastric emptying and small intestinal transit were measured by
evaluating the intestinal location of Rhodamine B-conjugated dextran (D1841,
Molecular Probes) as described elsewhere^{32 (link)} with modification. Six-week-old mice were given
2.5mg/ml of Rhodamine B-conjugated dextran in 5% methylcellulose (M7027, Sigma)
via gavage. Ten minutes after administration for the gastric emptying assay and
60 minutes after administration for the small intestinal transit assay, the
entire small bowel (unflushed) was divided into 10 equal segments and placed
into tubes with 4ml of PBS. The stomach was prepared similarly. Tissues were
homogenized and were centrifuged at 2,000 rpm for ten minutes to separate out a
pellet and supernatant. The fluorescence in each aliquot of the cleared
supernatant was read by a Synergy4 plate reader (excitation 540 nm/emission 625
nm, BioTek). Gastric emptying was calculated as the ratio of the total
fluorescent intensity in small intestine divided by total fluorescent intensity
in stomach and small intestine. For small intestinal transit, fluorescent
intensity for each small intestinal segment (I₁ to
I_10, numbered from oral side) was used to
calculate the geometric center of delivered dextran by the formula
below^{32 (link)}. $$geometriccenter=\sum _{n=1}^{10}\frac{I_n\times n}{I_1+I_2+\cdots +I_{10}}$$