5427r centrifuge

Fresh placenta of normally parturated, healthy Holstein cows, aged 3–5 years, weighing above 600 kg and parity 2–4, was collected from a dairy farm in Sichuan Province, China. Then immediately washed with normal saline to remove residual blood and dirt in the placenta and stored at −20 °C.
Preparation of CPE according to our previous method [6 (link)]. The placenta was thawed at room temperature and minced. Then, the placenta was homogenized in deionized water by a homogenizer (FSH 2A homogenizer; Yuexin, China) in an ice bath to obtain placenta homogenate. Next, hydrolyzed placenta homogenate by papain (800 U/mg, Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China) at neutral pH and 55 °C with 35.74% substrate concentration and 3.92% enzyme-substrate ratio for 5.49 h. Inactivate papain at 90 °C for 10 min, then centrifuged (5427 R centrifuge, Eppendorf, Hamburg, Germany) the solution at 9200× g for 5 min and collected the supernatant. Finally, the supernatant was freeze-dried for 48 h to prepare CPE (LyoQuest-55, Telstar, Terrassa, Barcelona).

Pure bacterial and fungal isolation was carried out according to the methods described by Gatheru et al. (2019 (link)). From each pure bacterial isolate plate, individual bacterial colonies were picked and then grown in Nutrient Broth (Oxoid, UK) for 24 h at 32 ± 2°C in a MIR-554 cooled incubator (PHC Europe B.V). From this bacterial broth, 1.5 mL was collected, and the bacterial cells were harvested through centrifugation (5427R centrifuge, Eppendorf, Hamburg, Germany) at 8,000 rcf for 5 min at 4°C. Genomic bacterial DNA extraction was done using the Isolate II genomic DNA extraction kit (Meridian Bioscience, London, UK) as per the manufacturer's instructions. On the other hand, fungi spores/mycelia were scrapped off from pure fungal culture plates. The spores/mycelia were collected in 2 mL Eppendorf tubes and genomic fungal DNA was extracted using the Isolate II Plant DNA Extraction Kit (Meridian Bioscience) following the manufacturer's instructions. The extracted bacteria and fungi DNA was quantified using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, USA). Bacteria and fungi DNA samples were stored at −20°C awaiting Polymerase Chain Reaction (PCR) analysis.

Brain tissue for protein isolation was obtained from 5 intact rats (untreated), 10 EAE rats (5 EAE 20d and 5 EAE 30d), and 10 EAE rats treated with olive leaf therapy (5 EAE+TOL 20d and 5 EAE+TOL 30d). The rat brain hemisphere samples were rapidly removed and snap-frozen in liquid nitrogen for protein isolation and stored at -80°C. Bead Ruptor 12 homogenizer (Omni International, Kennesaw GA, USA) was used for the preparation of brain tissue homogenates (10% w/v) from frozen samples. Briefly, one rat cerebral hemisphere was placed into 7 mL tube containing 1.4 mm ceramic beads and suspended with 100 mM Tris-HCl buffer pH 7.6 containing phosphatase and protease inhibitors. The samples were processed at speed 4 for three cycles; each was 15 sec with Dwell of 30 sec. The homogenates were then centrifuged in an Eppendorf 5427R centrifuge (Eppendorf, Hamburg, Germany) for 10 min at 5000 rpm and 4°C. The obtained supernatants were aliquoted and stored at -80°C until analysis. Protein concentrations in supernatants of brain homogenate were determined according to the manufacturer procedure using a BCA protein assay kit (Pierce, Thermo Scientific, Rockford, IL, USA).

An Agilent C18 column (Agilent Technologies, Inc., Santa Clara, CA, USA), 250 mm x 4.6 mm, with a particle size of 5 μm was used for high performance liquid chromatography (HPLC) (SHIMADZU LC-15C, Kyoto, Japan). The mobile phase contained 1% acetic acid and methanol at a ratio of 50:50 (v:v). The flow rate, injection volume, detection wavelength and column temperature were 0.6 mL/min, 20 μL, 260 nm and 30°C, respectively. In preparation for HPLC analysis, 2 mL of culture samples were collected and prepared by centrifuging (8,000×g) (Eppendorf 5427 R Centrifuge, Eppendorf AG, Hamburg, Germany) and filtering through 0.22-μm pore-size PTFE membrane syringe filters. Quantities of the analytes were examined from a standard curve prepared using standard 3MI (Shanghai Macklin Biochemical Co., Ltd, Shanghai, China). The quantity of 3MI in each sample was determined in mg/kg. The recovered quantity was also calculated as the percentage value with respect to the total concentration of 3MI added in the medium.

A constant-temperature incubator was purchased from Shanghai Shcimo Medical Device Manufacturing Co., Ltd; a high-speed refrigerated centrifuge was provided by Shanghai Anting Device Manufacturing Co., Ltd; a Dk-8D digital thermostatic laboratory water bath was from Guangzhou Hezhong Biotechnology Co., Ltd; a three-dimensional autoclave was obtained from Shanghai Shenan Medical Device Manufacturing Co., Ltd; 752 UV-VIS spectrophotometer was from Shanghai Jinghua Instruments; the biological purification table was from Suzhou Purification Equipment Co., Ltd; PacBio Single Molecule, real-time (SMRT) sequencing system was acquired from Pacific Biosciences of California; the Novaseq 6000 system was from Illumina; a 2100 Bioanalyzer Instrument was from Agilent Technologies; the Eppendorf 5427R centrifuge was from Eppendorf; the NanoDrop 2000 spectrophotometer was obtained from Thermal Fisher Scientific; the DYY-6C agarose gel electrophoresis kit was from Beijing Liuyi Biotechnology Co., Ltd; and finally, the PCR instrument was from Dongsheng Xingye Scientific Instrument Co., Ltd.

SpectraMax i3x multi-mode microplate reader (Molecular Devices, San Jose, CA, USA) was used for the fluorescence intensity analysis. Eppendorf centrifuge 5427R (Eppendorf, Hamburg, Germany) was used to separate samples. The black 96-well plates were purchased from Haimen Huabo experimental equipment Factory (Nantong, China). The ultrapure water produced by Medium-E400UP ultra-pure water purifier (HHitech, Shanghai, China) was used to prepare all solutions in this experiment.