High speed centrifuge

The following instruments and reagents were used in this experiment: NexION 300D ICP-MS (PerkinElmer, Waltham, MA, USA) equipped with a concentric nebulizer and collision reaction cell; Series 200 HPLC (Perkin Elmer, Waltham, MA, USA); de-iron water was acquired using a Millipore water purifier (Millipore Ltd., Bedford, MA, USA); Phenomenex Luna 5 μm C18 (250 × 4.6 mm) column; ETH031 Microwave Digestion Instrument (Milestone Co., Roseland, NJ, USA); KQ-500DE Numerical Control Ultrasonic Cleaner (Kunshan Instrument Co., Shanghai, China); high-speed centrifuge (Eppendorf Co., Hamburg, Germany); cobalt standard solution (1000 mg/L) (SPEX Co., Metuchen, NJ, USA); vitamin B12 (Cyanobalamin, Sigma-Aldrich Co., Burlington, MA, USA); infant milk powder standard substances GBW 10277 (NIM, Beijing, China); nitric acid and methanol (chromatographic purity, Merck, Darmstadt, Germany); ethylenediamine tetraacetic acid disodium (EDTA), potassium dihydrogen phosphate, disodium hydrogen phosphate dodecahydrate, potassium ferrocyanide, anhydrous sodium acetate, trichloroacetic acid, zinc acetate, and acetic acid (analytical purity, Chinese Medicine Co., Beijing, China).

The test animals were selected from a sizeable intensive dairy farm in Daqing, Heilongjiang Province, China. Three healthy 2-year-old cows of similar body condition and at mid-lactation were randomly selected. All cows were healthy and did not exhibit any clinical signs of mastitis. Whole blood was collected from the cows via the middle tail vein. Neutrophils were isolated and purified by using the bovine peripheral blood neutrophil purification kit^® (Solarbio, Beijing, China). Then, 4 mL Separate A, 2 mL Separate C, and 2 mL sodium citrate anticoagulated whole blood were successively added into a 15 mL centrifuge tube, forming a liquid gradient. The mixture was then centrifuged at room temperature in an Eppendorf high-speed centrifuge at 900× g for 30 min. After the centrifugation step, neutrophils were found in the lower gradient phase as a white ring. Neutrophils were extracted into a new 15 mL centrifuge tube and erythrocytes were lysed with Red Blood Cell Lysis Buffer. Then, the suspensions were centrifuged (300× g, 5 min) and washed three times; neutrophils were resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco Grand Island, NY, USA).

Blood was collected by tail vein puncture under aseptic conditions into a blood collection tube containing sodium citrate, and PMN was isolated using the Solarbio Bovine Peripheral Blood Neutrophil Isolation Kit^® (Solarbio, Bejing, China) according to the manufacturer’s instructions. First, 4 mL of Reagent A was added into a 15 mL centrifuge tube, then 2 mL of Reagent C was carefully added onto the surface of Reagent A, and finally, 2 mL of peripheral blood was added into the solution. In order to achieve cell separation, the solution was centrifuged in an Eppendorf high-speed centrifuge at 900× g for 30 min (Eppendorf, Hamburg, Germany). The granule cell layer was aspirated, added with RPMI 1640 medium, and washed 3 times by centrifugation at 500× g for 5 min. Trypan blue staining was used to determine cell death with the number of trypan-blue-positive and trypan-blue- negative PMN counted by an automatic cell counter, and its concentration was adjusted to 1 × 10⁶ cells/mL.

All the instrumentation used in this experiment included a double single-side purification workbench (Shanghai Shuo Guang Electronic Technology Corporation, Shanghai, China), Intelligent Biochemical Incubator (Shanghai Shuo Guang Electronic Technology Corporation, Shanghai, China), autoclave (Panasonic Healthcare Corporation, Tokyo, Japan), high-speed centrifuge (Eppendorf, Hamburg, Germany), Large-Capacity Combined Shock Incubator (Shanghai Minquan Instrument Corporation, Shanghai, China), FLEXA Purification System (Tianjin, China, Agela Technologies), Rotary Evaporator (Ningbo Hangjing Biotechnology Corporation, Ningbo, China), Waters Xevo G2-XS Q-Tof mass spectrometer (Waters Corporation, Milford, MA, USA), Agilent HPLC 1260 Infinity instrument (Agilent Technologies Inc., Santa Clara, CA, USA), and 600 MHz Bruker AVANCE NEO spectrometer (Bruker, Karlsruhe, Germany).

Fruit and vegetable samples were treated according to their water content. If the water content was >30% of fresh weight, fruits and vegetables were homogenized with a homogenizer. The homogenates were centrifuged for 15 min at 2000× g, in a laboratory centrifuge (Heraeus), and the clear supernatant was used for analysis. With insufficient phase separation, turbid supernatants were centrifuged at 8000× g for 4 min, in a high-speed centrifuge (Eppendorf, Hamburg, Germany), and the clear supernatants were analysed for polyphenols.
Fruits and vegetables with low water content (<30%) were weighed, and distilled water was added to the weight (mL/g) before homogenization, e.g., if a sample weighed 5.6 g, we added 5.6 mL of distilled water. For dry samples (e.g., flour), we added the 10-fold amount of water. The remaining procedure was as described before.