Fascalibur flow cytometer

Apoptotic cells in the populations were measured with a FACScan flow cytometer (Becton-Dickinson) by the AnnexinV Fluos apoptosis detection kit (Roche Molecular Biochemicals, Mannheim, Germany). Cells were stained with Annexin-V-FITC for exposure of phosphatidylserine on the cell surface as an indicator of apoptosis, following the manufacturer’s instruction (BD Biosciences). Data acquisition and analysis were performed using a BD Biosciences FASCalibur flow cytometer with CellQuest software. Positively stained by annexin-V-FITC only (early apoptosis) and propidiumiodide (late apoptosis) were quantitated, and both subpopulations were considered as overall apoptotic cells.

Single cell suspension from BM was prepared as previously described (16 (link)). 1 x 10⁶ cells were blocked with anti-mouse CD32/CD16 monoclonal antibody (BD Biosciences) and then stained with fluorescein isothiocyanate (FITC)-conjugated anti–mouse Ly6G antibody (clone 1A8, BD Biosciences); phycoerythrin (PE)-conjugated anti–mouse Gr-1 antibody (clone RB6-8C5, BD Biosciences), biotinylated-conjugated anti-mouse Ly6C antibody (clone AL-21, DB Biosciences); FICT-conjugated anti-mouse CD34 antibody (clone RAM34, BD Biosciences); biotinylated-conjugated anti-mouse Ly 6A/E antibody (Sca-1, clone D7, BD Biosciences); PE-conjugated anti-mouse CD117 antibody (C-kit, clone 2B8, DB Biosciences); FITC-conjugated anti-mouse TER-119/Erythroid cells antibody (clone Terr-119, BD Biosciences); and allophycocyanin (APC) mouse lineage antibody cocktail (clone 145-2C11). Following incubation with biotinylated primary antibodies, the labeling was revealed using streptavidin-PercP. The cells were acquired on a FASCalibur™ flow cytometer (BD Biosciences) and data were analyzed with FlowJo software (Tree Star).

The secretion of ROS by NCTC-1469 cells was tested using 2, 7-dichlorofluorescein diacetate (DCFH-DA) fluorescence assay [31 (link),33 (link)]. NCTC-1469 cells (1 × 10⁵ cell/well, 200 μL/well) were seeded in 96-well plate and incubated with MBPHs-I (0.1, 0.2, 0.4mg/mL) for 24 h and then transferred to a EP tube (microcentrifuge tube for separation of trace reagents) of 1.5 mL, which was centrifuged using at 1500× g for 5 min. The supernatant was removed and the cells were washed using PBS for twice, then 100 μL of PBS was added aimed to resuspend cells. Aliquot of 10 μM DCFH-DA fluorescent probe solution was added in three groups and the resulting mixture was incubated for 0.5 h at 37 °C. After washing twice with PBS to remove the extracellular DCFH-DA, the cells were resuspended into a single cell suspension. Finally, the intracellular ROS level was determined with a BD FASCalibur flow cytometer (Franklin Lakes, NJ, USA).

RAW264.7 cells were seeded in six-well plates at a density of 1 × 10⁶ cells/well. After 4 h of incubation, the culture medium was removed, and the cells were treated with 2 mL of culture medium containing 1 μg/mL LPS (final concentration, model group), or 2 mL of culture medium containing 1 μg/mL LPS (final concentration) and phytosterols at different concentrations (25, 50, 100, and 200 μM, experimental groups). Meanwhile, 2 mL of culture medium without phytosterols and LPS was added as the control group. After incubation for 24 h, the culture medium was removed, and the cells were washed two times with PBS; then, 100 μL of FITC-dextran (1 mg/mL) was added, and the cells were covered with tin foil paper to protect from light and incubated at 37 °C for 1 h. After incubation, 1 mL of PBS was added. Then, RAW264.7 cells were harvested and washed three times with PBS, before being resuspended in 500 μL of PBS buffer. The stained cells were then analyzed by a BD FASCalibur flow cytometer (Franklin Lakes, NJ, USA) [35 (link),36 (link)].

Following trypsin digestion, 1×10⁶ MG-63 cells were harvested, washed twice with ice-cold phosphate-buffered saline (PBS), fixed and permeabilized with 70% ethanol at −20°C for 24 h, and washed once with ice-cold PBS. After incubation with propidium iodide (PI) staining buffer at 37°C for 1 h, the cells were washed one more time with ice-cold PBS and DNA content analysis was performed with a FASCalibur Flow Cytometer (Becton, Dickinson and Company). The PI staining buffer contained 1X PBS, 100 μg/μl RNase and 40 μg/ml PI.

Samples were taken from the initial microbial inocula (day 0) and all microcosms (day 5) for determinations of microbial cell abundances. For bacterial abundance, 4 mL were preserved with formaldehyde at a final concentration of 2%, and immediately flash frozen in liquid nitrogen until used in the flow cytometry measurement. Cells in the samples were stained using 10x SYBR Green I (Life Technologies, Darmstadt, Germany) and then counted in a FASCalibur flow cytometer (Becton Dickinson, Fremont, CA, United States), as described elsewhere (Gasol and del Giorgio, 2000 (link)).
In addition, the cell abundance of protists was determined using epifluorescence microscopy, according to Weber et al. (2012) (link) with minor modifications. Briefly, 10 mL of sample was fixed with formaldehyde at a final concentration of 2% and stored at 4°C. After ∼4 h, the fixed samples were filtered onto 0.8-μm, 25-mm black filters (Whatman, Dassel, Germany), which were then stored at −20°C until further processing. For cell enumeration, cells on the filters were stained with 4’,6-diamidin-2-phenylindol and three randomly selected fields of view were inspected at 63× magnification using a Zeiss Axioskop 2 mot plus microscope (Zeiss, Oberkochen, Germany). Technical triplicates were established for each sample.