Adp assay kit

Isolated colon mitochondria were homogenized in 9 volumes of 100 mM Tris and 4 mM EDTA buffer, pH 7.75, preheated to 95 °C. Samples were incubated at 100 °C for 2 min and centrifuged at 1000×g for 2 min at 4 °C. The supernatant (50 µl) was used to determine ATP with 1 volume of reaction buffer. Luminescence was measured in 96-well plates with FLUOstar Omega (BMG Labtech) using ATP Bioluminescence Assay Kit HS II (Roche, 11699695001) following the manufacturer’s instructions.
Proteins from isolated colon mitochondrial were precipitated in 6 volumes of 6% perchloric acid and neutralized with KOH for the determination of ADP and AMP using ADP Assay Kit (MilliporeSigma, MAK133) or AMP Assay Kit (Abcam, ab273275), respectively, following the manufacturer’s instructions. For determination of total ADP and ATP as well as AMP in the cells, the same ADP Assay Kit (MilliporeSigma, MAK133) or AMP Assay Kit (Abcam, ab273275), respectively, were used. Luminescence and absorbance were measured in 96-well plates with FLUOstar Omega (BMG Labtech).
The adenosine released into the cell culture medium at 24 h lines was determined using the fluorometric Adenosine Assay Kit (Abcam, ab211094) following the manufacturer’s instructions. Fluorescence was measured in 96-well plates with FLUOstar Omega (BMG Labtech).

The concentrations of ADP (Selleckchem, catalog S9368) in macrophages were measured by ADP Assay Kit (MilliporeSigma, catalog MAK133) following the instructions. The process is outlined as follows. A total of 1 × 10⁴ PMAs were directly cultured in the assay microplate and stimulated by LPS or ADP for 24 hours. Working agents were used to lysis cells to release ATP and ADP. In the presence of fluorescein, ATP immediately reacts with the substrate (D-fluorescein) to produce luminescence. ADP was converted to ATP by an enzyme reaction, and this newly formed ATP then reacted with D-luciferin in the previous step. The luminescence (relative light units) was read by a luminometer immediately, using the value obtained from the appropriate standards to plot a standard curve and determine the amount of ADP present in the samples. Mice with colitis in the NC and the ADP groups were all constructed by 3% DSS. The ADP (250 mg/kg) was i.p. injected into the ADP group, while mice from the control group were i.p. injected with the same amount of saline every day for 7 days.

The concentrations of ADP (Selleckchem, catalog S9368) in macrophages were measured by ADP Assay Kit (MilliporeSigma, catalog MAK133) following the instructions. The process is outlined as follows. A total of 1 × 10⁴ PMAs were directly cultured in the assay microplate and stimulated by LPS or ADP for 24 hours. Working agents were used to lysis cells to release ATP and ADP. In the presence of fluorescein, ATP immediately reacts with the substrate (D-fluorescein) to produce luminescence. ADP was converted to ATP by an enzyme reaction, and this newly formed ATP then reacted with D-luciferin in the previous step. The luminescence (relative light units) was read by a luminometer immediately, using the value obtained from the appropriate standards to plot a standard curve and determine the amount of ADP present in the samples. Mice with colitis in the NC and the ADP groups were all constructed by 3% DSS. The ADP (250 mg/kg) was i.p. injected into the ADP group, while mice from the control group were i.p. injected with the same amount of saline every day for 7 days.