Ip3 elisa kit

The levels of IP3 were determined using an IP3 ELISA kit (Cusabio Biotech Co., Ltd., Wuhan, China) according to the manufacturer’s instructions. Briefly, the transverse colons (100 mg) were washed and homogenized in ice-cold PBS (pH 7.2–7.4) with a glass homogenizer (Sigma-Aldrich Co.). The tissue lysates were then centrifuged at 1,000 rpm for 5 min at room temperature, after which the supernatant was collected for analysis. An anti-IP3 detection antibody was added and incubated at 37°C for 60 min, after which substrate solution was added and incubated for 15 min at 37°C. The reaction was terminated following the addition of stop solution and the plates were read at an absorbance of 450 nm using a Molecular Devices VERSA max Plate reader (Sunnyvale, CA, USA).

Levels of IP3 were determined using an IP3 ELISA kit (Cusabio Biotech Co., Ltd., Wuhan, China), according to the manufacturer’s instructions. The frozen transverse colon tissue was washed and homogenized in ice-cold 1X PBS (pH 7.2–7.4) using a glass homogenizer (Sigma-Aldrich Co., St. Louis, MO). The tissue lysates were centrifuged at 1000 rpm for 5 min at room temperature, after which the supernatant was collected for analysis. An anti-IP3 detection antibody was added and incubated at 37 °C for 60 min, after which the substrate solution was added and the samples were further incubated for 15 min at 37 °C. The reaction was terminated following the addition of stop solution, and the plates were read at an absorbance of 450 nm using a Molecular Devices VERSA max Plate reader (Molecular Devices, Sunnyvale, CA).

HAECs were plated in 96-well culture plates at a density of 1 × 10⁴ cells/well and cultured overnight. Culture media were replaced with 250 μL of EGM-2 containing test drugs and incubated for 1 h. Supernatants were collected and stored at –80°C until further analysis. IP3 was assessed using human inositol 1,4,5-trisphosphate, IP3 ELISA Kit (Cusabio, Wuhan, China) according to the manufacturer’s instructions. Optical density was measured at 450 nm using a microplate reader (Soft max, Molecular Devices). Results are expressed as IP3 concentration (pg/mL).

The levels of IP₃ were determined using an IP3 ELISA kit (Cusabio Biotech Co., Ltd, Wuhan, China), according to the manufacturer’s instructions. The ASMC culture medium was removed and the cells were incubated with 0.1 mmol/l HClO₄ for 20 min. The cells were centrifuged at 170 × g for 15 min at room temperature and the supernatant was collected for analysis. An anti-IP₃ detection antibody was added and incubated at 37°C for 60 min, followed by the addition of substrate solution for 15 min at 37°C. The reaction was terminated following the addition of stop solution and the plates were read at an absorbance of 450 nm using a Model 680 spectrophotometer (Bio-Rad Laboratories, Inc.). The effect of formoterol on the expression of IP₃ was determined using the following formula: Inhibition of ACh-induced IP₃ accumulation (%) = (IP₃ levels in the control group - IP₃ levels in the treatment group) / IP₃ levels in the control group × 100%.

Levels of IP3 were determined using an IP3 ELISA kit (Cusabio Biotech Co., Ltd., Wuhan, China), according to the manufacturer’s instructions. Briefly, the frozen colon tissues were washed and homogenized in ice-cold PBS (pH 7.2–7.4) using a glass homogenizer (Sigma-Aldrich Co.). Tissue lysates were centrifuged at 1000 rpm for 5 min at room temperature, after which the supernatant was collected for analysis. An anti-IP3 detection antibody was added and incubated at 37 °C for 60 min, after which the substrate solution was added and the samples were further incubated for 15 min at 37 °C. The reaction was terminated following the addition of stop solution, and the plates were read at an absorbance of 450 nm using a Molecular Devices VersaMax Plate Reader (Molecular Devices, Sunnyvale, CA, USA).