Ethics statement
All animal experiments were performed according to the guidelines of the animal ethical organization and obtained the permission of Shanghai Gongli Hospital, the Second Military Medical University
Materials and reagents
Lipopolysaccharides from Escherichia coli 0111:B4 (catlog: L2630, sigma), antibiotic-Cefuroxime Axetil (Chengdu beite pharmaceutical co. LTD),
Total Exosome Isolation Reagent (catlog: 4478359, Invitrogen™), ExoAb Antibody Kit (catlog: EXOAB-KIT-1, SBI), anti-mouse Galectin 9 (catlog: ab69630, abcam),
Transmission Electron Microscope (catlog: HT7700, HITACHI), Mouse lymphocyte isolation medium (catlog: LTS1092, shanghai yanjin bio. co. ltd), RPMI-1640 medium (Thermo Fisher Scientific, USA), Fetal Bovine Serum (gibico), Fixation/Permeabilization Solution (catlog: 554722, BD ), Wash Buffer (catlog: 554723, BD), Flow Cytometry Staining Buffer (catlog: 00-4222-57, eBioscience™), anti-Mouse CD16/CD32 (catlog: MFCR00, Invitrogen), anti-mouse CD4-FITC (catlog: 11-0040-85, eBioscience™), anti-mouse IFN-γ-PE (catlog: 12-7319-42, eBioscience™), anti-mouse IL-17-PE-Cy7 (catlog: 25-7042-82, eBioscience™), anti-mouse CD25-APC (catlog: RM6005, Invitrogen), anti-mouse Foxp3-PE-Cy5.5 (catlog: 35-4776-41, eBioscience™), Mouse IL-1β ELISA Kit (catlog: 70-EK212/3-96, MultiSciences), Mouse IL-6 ELISA Kit (catlog: 70-EK206/3-96, MultiSciences), Mouse IL-10 ELISA Kit (catlog: 70-EK210/3-96, MultiSciences), Mouse IFN-γ High Sensitivity ELISA Kit (catlog: 70-EK280HS-96, MultiSciences)
Infectious severe sepsis mice model establishment and FFP transfusion
The forty number C57/BL6 mice were brought from Shanghai bangyao biotechnology co. LTD(No.A0001), 10-12 weeks old, they were maintained in specific-pathogen-free conditions in the animal care facility, after one week adapted cultivation, ten mice were anesthetized with 2% isoflurane and sacrificed, then collected the blood samples from the mice aorta abdominalis immediately for FFP transfusion, while, the other thirty mice were
administered with Escherichia coli 0111:B4 (100×106 colony forming units [CFUs] by intraperitoneal injection. Besides, the methods of bacteria cultivation were listed below21: the Escherichia coli 0111:B4 were stored in 20% glycerol at -70℃, then, obtained some bacteria in frozen with inoculation loops and cultured them on LB agar (sigma) for 12 hours (h), 37℃, afterwards, the colonies were counted with scratch inoculation method after overnight incubation. Thereafter, judging from the mice breath, mobility and food/water intake to estimate endotoxemia three times daily for up to seven days, while, the mice appeared with tremble, high fever and difficult breathing, they were estimated severe sepsis. Then, the infectious sepsis mice model were all gavaged antibiotic-Cefuroxime Axetil (75mg/d, per mice) and randomly divided into two groups, one group mice were received FFP transfusion via the lateral tail vein, while, the other group model mice were received equivalent saline injection. After FFP transfusion and the mice recovered from endotoxemia, the mice were anesthetized with 2% isoflurane and sacrificed, then collected the blood samples from all the mice aorta abdominalis immediately.
Exosome isolation and identification
The collected blood samples of mice were centrifugated at 3000g/min to obtain the serum, then the Total Exosome Isolation Reagent were added to the serum with reverse blending and placed it for one night, 4 ℃, afterwards, the mixed medium were centrifuged with 3000g/min, 5 min for three times, while, the precipitates were the exosome, the precipitates in three tubes were extracted the proteins, then analyzed the marker proteins CD63, CD81 with ExoAb Antibody Kit, besides, the exosome protein Galectin 9 was also detected with western blot. While, the precipitates in other tubes were resuspended with PBS, then visualized it with Transmission Electron Microscope.
Flow cytometry
The lymphocyte were isolated from the collected blood samples of mice with mouse lymphocyte isolation medium at 3000g/min centrifugation. Then, the obtained lymphocytes were cultured in RPMI-1640 medium with FBS and counted. Later,
3-5×106 cells were put in each tube and they were permeabilized with Fixation/Permeabilization Solution for 30 min away from light, then, the anti-Mouse CD16/CD32 (1μg/106 cell) were added in each tube for 30 min, 4 ℃ away from light.
Afterwards, the cells were washed with wash buffer, and one tube wasn’t received antibody incubation, the other tubes were dividedly received double staining antibodies dilution buffer: anti-mouse CD4-FITC mixed with anti-mouse IFN-γ-PE, anti-mouse CD4-FITC mixed with anti-mouse IL-17-PE-Cy7, anti-mouse CD25-APC mixed with anti-mouse Foxp3-PE-Cy5.5 for 30 min, 4 ℃ incubation away from light, Thereafter, the cells were washed twice and re-suspended in Flow Cytometry Staining Buffer for Flow Cytometry analysis with instrument Calibur FACS (Bioscience, BD, USA).
Elisa assay
The collected blood samples of mice were centrifugated at 3000g/min to obtain the plasma for three times, then the plasma was analyzed the production of cytokines IL-1β, IL-6, IL-10 and IFN-γ with the enzyme linked immunosorbent assay (ELISA) kits, finally, the microplate reader (Bio-Tek, Epoch) was used to measure optical density at 450 nm, all the procedures were accorded with the reagents manufacturers’ instructions.
Statistical analysis
The data were analyzed with one-way ANOVA method in SPSS19.0 software. The significance was defined as P<0.05. All graphs were depicted with Graphpad prism 6.0 presenting data with mean ± SEM. Data are representative of at least two or three independent experiments.