Protease inhibitor cocktail 1

The uterine tissue was stored at −80 °C and was weighed at 0.1 g into a 2-mL tube, mixed with 500 μL of RIPA lysis buffer (Solarbio, Peking, China) containing 1% protease inhibitor cocktail I (Medchem express, Monmouth, NJ, USA), and ground with a tissue homogenizer. Grinding was performed until no intact tissue fragments were visible, and all steps of the grinding process were performed on ice. The supernatant was collected, centrifuged, and stored in a −20 °C refrigerator (15,000× g, 4 °C, and 10 min). The levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were measured in the supernatant of uterine tissue homogenates using ELISA kits (Jinma, Shanghai, China) according to the manufacturer’s instructions. Absorbance was obtained at 450 nm using a Multiskan™ GO full-wavelength microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

The levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), interleukin-18 (IL-18), and tumor necrosis factor-α (TNF-α) (Jinma Co., Ltd., Shanghai, China) were measured in the supernatant of uterine tissue homogenates and the supernatant of the BEECs (stimulated by NETs) using ELISA kits (Jinma, Shanghai, China) according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a Multiskan™ GO full-wavelength microplate reader (Thermo Fisher Scientific, Rockford, IL, USA). First, the uterine tissue, stored at −80 °C, was weighed at 0.1 g in a 2 mL tube, mixed with 500 µL of RIPA lysis buffer (Solarbio, Beijing, China) containing 1% protease inhibitor cocktail I (Medchem Express, Monmouth Junction, New Jersey, USA), and ground with a tissue homogenizer. Grinding was performed until no intact tissue fragments were visible; all steps of the grinding process were performed on ice. The supernatant was collected, centrifuged, and stored in a −20 °C refrigerator (15,000× g, 4 °C, and 10 min).