Multiskan go full wavelength microplate reader

A series of main instrumentations were used in this study, such as tissue scissors, hemostatic clamps (Shanghai surgical instrument factory), an AEG-220 type electronic analytical balance (Shimadzu, Japan), vortex mixing apparatus Haimen Qilin Bell Instrument Manufacturing Co., Ltd. Model XW-80A), water bath nitrogen blowing instrument (Beijing Cheng Meng Albert Technology Co., Ltd., model CW-12) and KQ-500DE CNC ultrasonic cleaner (Kunshan City ultrasound Instrument Co., Ltd.). HPLC-HR-MS system was consists of several units: Thermo Accela Ultra-High Performance Liquid Chromatograph (Accela 600 Pump with Accela Open Autosampler and Quaternary Solvent Controller), Thermo LQT Orbitrap XL Mass Spectrometer, Xcalibur Workstation, ultra-pure helium as collision gas and high purity nitrogen for atomization. Structures were confirmed by AM-500 nuclear magnetic resonance instrument (Switzerland Bruker Company). In addition, Forma 3111 CO₂ incubator, Multiskan GO full-wavelength microplate reader (Thermo Fisher, American), inverted microscope (OlympusIX71), and biological safety cabinet (HF) were used to finish the active test experiment.

We employed an MTT kit (Thermo Fisher Scientific Co., Ltd., Hangzhou, China) to test the PC12 cell proliferation activity. Cells were incubated at 37°C with 5% CO₂ for 24 h, 48 h, 72 h, and 96 h. Then, the medium was discarded and 20 μL of MTT was added to per well for incubation for 4 hours. Next, we added 150 μl of dimethyl sulfoxide to the well and shook the plate for 5–10 minutes to make sure the purple crystals were completely dissolved. Multiskan™ GO full-wavelength microplate reader (Thermo Fisher Scientific Co., Ltd., Hangzhou, China) was employed to test the absorbance at 450 nm to detect cell proliferation. The experiment was repeated 3 times.

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).

The strains were inoculated into Tiecha-free liquid medium and cultured in a constant temperature shaker (HS-200B, Huanan Xinhai, Shenzhen, China) at 28 °C with 150 r/min for 48 h. After the culture, 2 to 5 mL of culture medium was absorbed and filtered by 0.22 μm sterile filter membrane, and an equal volume of CAS detection solution was added. After standing for 1 h, full-wavelength enzyme labeler (model: Multiska GO) was used to measure OD 630 (denoted as “AS”), and OD630 of uninoculated liquid medium (denoted as “Ar”) was measured by the same method. The concentration of siderophore was expressed by siderophore unit (SU): SU = [(AR − AS)/Ar] × 100%. The determination was repeated 3 times, and the mean value was taken for comparative analysis.
The structure type of siderophore in the above-mentioned culture liquid was determined by the method of the FeCl₃ test [24 (link)]. The spectrophotometric data were recorded on a Multiskan GO full-wavelength microplate reader (Thermo Scientific, Waltham, MA, USA). Ferric hydroxamate absorbs light at 420–450 nm, while a 495 nm peak of ferrated siderophore indicates the presence of a catecholate siderophore.

The instruments used were as follows: SHIMADZU Nexera X2 UPLC (Kyoto, Shimadzu, Japan), Applied Biosystems 4500 QTRAP mass spectrometer (Applied Biosystems, Waltham, MA, USA), MULTISKAN GO full-wavelength microplate reader (Thermo Fisher, Waltham, MA, USA), 3K15 centrifuge (Sigma-Aldrich, St. Louis, MO, USA), WP-UP-YJ-20 micro-organic heat removal ultra-pure water machine (Sichuan Water Treatment Equipment Co., Chengdu, China), and ME204E electronic balance (Shanghai Mettler-Toledo Instruments, Shanghai, China). The reagents used were as follows: 2,2-biphenyl-1-bitter hydrazinyl (Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai, China), 2,2′-Hydrazinyl-bis(3-ethylbenzothiazoline-6-sulphonic acid) diamine salt (Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai, China), glucose (Sigma-Aldrich, St. Louis, MO, USA), anhydrous ethanol (analytical grade, Chengdu Kelong Chemical Reagent Factory, Chengdu, China), phenol (analytical grade, Chengdu Kelong Chemical Reagent Factory, Chengdu, China), concentrated sulphuric acid (analytical grade, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), acetonitrile (mass spectrometry grade, Merck & Co., Rahway, NJ, USA), and methanol (mass spectrometry grade, Merck & Co., Rahway, NJ, USA).