Production of reactive oxygen species (ROS) was detected using the non-fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). DCFH-DA diffused into cells and hydrolyzed into the 2′,7′-dichlorofluorescein (DCFH) which was hard to pass through the cytomembrane. Then DCFH reacted with ROS to form the fluorescent product DCF. Briefly, ARPE-19 cells and hRPE were transfected with p22phox siRNA and stimulated with Ang II as aforementioned. Then the medium was removed and cells were washed with ice-cold PBS. DCFH-DA (Beyotime, Shanghai, China) was diluted in fresh DMEM/F12 at a final concentration of 10 μM and incubated with cells for 30 min at 37 °C. After DCFH-DA treatment, the chemicals were removed and loaded cells were washed three times with PBS. The fluorescence was read at 488 nm excitation and 525 nm emission by the BD FACS Vantage SE Flow Cytometer (BD, Shanghai, China).
Facs vantage se flow cytometer
Manufactured by BD
Sourced in United States, Italy
The FACS Vantage SE flow cytometer is a laboratory instrument used for the analysis and sorting of cells or other particles. It employs fluidics, optics, and electronics to rapidly measure and analyze multiple physical and chemical characteristics of individual cells passing through a laser beam.
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Lab products found in correlation
Cellquest software, by BD (7 mentions)
Co2 incubator, by Thermo Fisher Scientific (6 mentions)
Dcfh da, by Beyotime (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit, by BD (2 mentions)
G44 26, by BD (2 mentions)
Tcs sp2 confocal laser scanning microscope, by Leica (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Facsdiva software, by BD (2 mentions)
Goat anti pax7, by LifeSpan BioSciences (1 mentions)
Rabbit anti pth1r, by Merck Group (1 mentions)
Alexa fluor 488 labeled donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Ribonuclease a, by Merck Group (1 mentions)
Magnetic bead, by Miltenyi Biotec (1 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dcfh da, by BD (1 mentions)
Dcfh da, by Merck Group (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
59 protocols using facs vantage se flow cytometer
1
ROS Detection in ARPE-19 Cells
2
Isolation and Characterization of Myogenic Cells
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The human myogenic cells were isolated, as previously described35 (link). The FACS analysis was performed using goat anti-Pax7 (LifeSpan BioSciences, Seattle, WA, USA), rabbit anti-PTH1R (Sigma-Aldrich), and PE-Cy7-labeled anti-human CD34 (BioLegend, San Diego, CA, USA) primary antibodies. Alexa-Fluor-488-labeled donkey anti-goat IgG (Life Technologies) and PE-labeled donkey anti-rabbit IgG (eBioscience, San Diego, CA, USA) antibodies were used as secondary antibodies for Pax7 and PTH1R detection, respectively. Flow cytometry and cell sorting were performed using a BD FACSVantage SE flow cytometer (BD Bioscience, San Jose, CA, USA) commission to ReproCell (Kanagawa, Japan).
3
Cell Cycle Analysis of Renca Cells
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Renca cells stimulated, as described above for the apoptosis assay for 48 h, were treated with trypsin, centrifuged at 800 rpm for 5 min at 37°C and fixed with pre-chilled 75% ethanol for >18 h at 4°C. Following treatment with 1% ribonuclease A (Sigma-Aldrich) for 30 min at 37°C, the cells were stained with 50 µg/ml PI for 30 min at 4°C. A BD FACSVantage SE flow cytometer was used to examine the cell cycle distribution at 490 nm. The proportion of cells in each cycle were analyzed using multicycle DNA content and CellQuest cell analysis software. Each experiment was performed in triplicate.
4
Isolation and analysis of human T cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from ethylenediaminetetraacetic acid blood in 2003 by standard-density centrifugation using Percoll (Pan-Biotech, Aidenbach, Germany) and double stained with mouse anti-human Vβ1 (clone BL37.2) and CD8+ (clone LT8). CD8+Vβ1+ double-positive T cells were sorted using a BD FACSVantage SE flow cytometer and analyzed by single-cell PCR. Cells were either sorted directly into PCR tubes or obtained as bulk and then isolated by hand under a microscope. CD8+ T cells from 2003 to 2005 were analyzed by complementarity determining region (CDR) 3 spectratyping, as described previously.6 (link)To isolate MAIT cells from PBMCs in 2013 and 2014, T cells were negatively isolated using the pan T-cell isolation kit (Miltenyi Biotec) and subsequently stained with the FITC-labeled mouse anti-human Vα7.2 (1:20, 3C10, BioLegend) and the allophycocyanin-labeled mouse anti-human CD161 (1:10, 191B8, Miltenyi Biotec) antibodies at 4°C for 30 minutes. Cells were then sorted using a BD FACSAriaTM2 flow cytometer. 5 × 102 CD161+Vα7.2+ cells, 6 × 103 CD161−Vα7.2+ cells, 1 × 105 CD161+ cells, and 2 × 105 CD161− cells were collected. CD4+ and CD8+ T cells were positively isolated from PBMCs in 2013 and 2014 using magnetic beads (Miltenyi Biotec).
5
Apoptosis and Cell Cycle Analysis
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Cellular apoptosis was analyzed using Annexin V and propidium iodide (PI) kit according to the manufacturer’s instructions. To determine the cell cycle assay, the treated cells were harvested, fixed overnight with 70% ethanol at 4 °C and incubated with PI staining reagent46 . Then the stained cells were subjected to a BD FACSVantage SE Flow Cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using Flow Jo 7.6.1 (Tree Star Inc., Ashland, OR, USA) and Modfit (BD Biosciences, USA) software.
6
Evaluating Cell Surface Markers
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To evaluate the surface expression of ALDH1, PD-L1, or anti-PD-L1 CAR, cells were washed with phosphate-buffered saline containing 2% bovine serum albumin (Sigma-Aldrich) and incubated with phycoerythrin- or allophycocyanin-labeled mouse anti-human antibodies (Cell Signaling Technology, Danvers, MA, USA). Isotype antibodies were used as control. The stained cells were evaluated using a BD FACS Vantage SE Flow Cytometer (BD Biosciences, USA), and the results were analyzed using the included software.
7
SPG-56 Induces Apoptosis in MCF-7 Cells
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MCF-7 cells (4 × 105 cells/well) were treated with various concentrations of SPG-56 (0, 10, 20 μg/ml) for 24 h. Cell apoptosis was detected using an Annexin V and propidium iodide (PI) kit (Wanleibio, Shenyang, China) according to the manufacturer’s instructions. The stained cells were subjected to a BD FacsVantage SE Flow Cytometer (BD Biosciences, San Jose, CA, USA). The data were analyzed using Flow Jo 7.6.1 software (Tree Star Inc., Ashland OR, USA).
8
ROS Generation in Neutrophils under Hyperglycemia
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The effect of high glucose on ROS generation was detected by 2′,7′-Dichlorofluorescein Diacetate (DCFH-DA, Sigma-Aldrich, St. Louis, MO, USA) and Dihydroethidium (DHE, BestBio, China). Before stimulation, neutrophils were preloaded with DCFH-DA and DHE (10 μM with 5.5 mM glucose DMEM) for 20 min. Next, we removed the medium and washed the loaded cells with PBS three times. Then, neutrophils were exposed to DMEM medium with different glucose concentrations (5.5, 15, 25, and 35 mM), and stimulation of each concentration lasted for 15 and 30, 60, and 120 min. Finally, the fluorescence signals of DCFH-DA was read at 488 nm excitation and 525 nm emission by the BD FACS Vantage SE Flow Cytometer (BD), and DHE was read at 535 nm excitation and 610 nm emission by a microplate reader (Thermo Fischer).
9
Annexin-V-FITC and PI Apoptosis Assay
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HEK293FT cells were collected by centrifugation at 200 × g for 5 min and resuspended in a binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2) at room temperature at a density of 1 × 106 cell/ml. The cells (100 μl) were mixed with 5 μl of annexin-V-FITC (BD Biosciences, Shanghai, China) and 5 μl of PI in a culture tube and incubated at room temperature in the dark for 15 min. After addition of 400 μl binding buffer, the cells (~10,000 cells per assay) were then analyzed by using a dual-laser FACS VantageSE flow cytometer (Becton Dickinson, Mountain View, CA) within one hour period. The percentages of apoptotic cells at early stage for each sample were estimated.
10
Fluorescence-activated Cell Sorting of Muscle Cell Subsets
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For fluorescence-activated cell sorting (FACS), the entire cell suspension obtained from collagenase digestion of tissue fragments (~5x106 cells/10x102 mg), was pelleted, resuspended and preincubated in PBS/1% BSA for 30 min on ice with regular mixing. After washing in PBS, cells were incubated for 30 minutes on ice with a phycoerythrin (PE)-conjugated anti-human CD146 monoclonal antibody for separation of cell subsets [25 (link)]. CD146+ cells were sorted using a FACSvantageSE flow cytometer (Becton Dickinson). ~5x104 (~1% of total nucleated cells) freshly vital CD146+ cells were harvested from the digestion of tissue fragments. For flow cytometry, the entire human freshly collagenase-released muscle cell suspensions before culture, were pre-incubated in PBS/1% BSA for 30 min, then for 30 min on ice with antibodies for analysis of cell subsets co-expressing CD146 along with N-CAM/CD56, a marker of satellite cells, Alkaline Phosphatase (ALP) and CD34 as a marker of endothelial cells (listed in S1 Table ). Expression of markers was assessed by using a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson Biosciences, San Diego, CA).
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