Intracellular ROS levels under normal and stress conditions were detected using a 2′,7′–DCFDA assay kit. The A2780/CP and A2780 cells were treated with 1 µg/mL PEI and 2.5 µg/mL CIS, as well as with PM/C and PMC complexes (at concentrations of 1 µg/mL PEI, 1 µg/mL MNP, and 2.5 µg/mL of CIS), compared to untreated cells, in the presence and absence of SMF for 48 h, which were prepared in RPMI medium supplemented with 10% FBS in 6-well cell culture plates (SPL Life Sciences Co., Ltd. Korea). After treatment, the cells were prepared immediately, according to the manufacturer’s instructions. Briefly, the cells were washed with PBS. The samples were then suspended in a conical test tube with 20 µM DCFDA in 1X buffer and incubated at 37°C in the dark for 30–45 min. ROS production was monitored immediately using a FACSCalibur Becton-Dickinson flow cytometer (Franklin Lakes, NJ). DCFDA flow cytometric data were analyzed using FlowJo software (version 7.6.1) (22 (link), 27 (link), 45 (link)).
Facs calibur becton dickinson flow cytometer
Manufactured by BD
Sourced in United States, Germany
The FACS Calibur Becton Dickinson flow cytometer is a laboratory instrument designed for the analysis and sorting of cells or particles suspended in a fluid stream. The core function of this device is to detect and measure physical and fluorescent characteristics of individual cells or particles as they pass through a laser beam.
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Lab products found in correlation
Dcfh da, by Serva Electrophoresis (3 mentions)
Propidium iodide, by Merck Group (3 mentions)
H2dcfda, by Thermo Fisher Scientific (2 mentions)
6 well cell culture plate, by SPL Life Sciences (1 mentions)
Cellquest program, by BD (1 mentions)
Rnasa, by Merck Group (1 mentions)
Lsrfortessa, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
Facscanto, by BD (1 mentions)
Fitc rabbit anti active caspase 3 antibody, by BD (1 mentions)
Rhodamine 123, by Thermo Fisher Scientific (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
14 protocols using facs calibur becton dickinson flow cytometer
1
Intracellular ROS Levels Under Stress
2
Cell Cycle Analysis by Flow Cytometry
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Cell suspensions were washed and incubated with 0.5 mL of PBS and 4.5 mL of ethanol 70% for 4 h at 4 °C. After 10 min and 310 g centrifugation, the cells were resuspended in 0.1% Triton X-100, 20 µg/mL propidium iodide (PI, Sigma-Aldrich, St. Louis, MO, USA), and 0.2 mg/mL of RNAsa (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37 °C for 30 min. PI fluorescence was detected by exciting the sample at 488 nm using a FACScalibur Becton Dickinson flow cytometer (Becton Dickinson, San Jose, CA, USA) with a 585/42 filter. The percentage of cells in each cell cycle phase was calculated using the CellQuest program (Becton Dickinson), using positive and negative controls and studying at least 104 cells in each sample: SubG1, G0/G1, S, and G2/M (fractions indicative of apoptosis, growth, DNA synthesis, and growth/mitosis, respectively).
3
Cell Cycle Analysis by Flow Cytometry
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Briefly, 2 × 105 cells/Petri dish (dimensions 60 × 15 mm, NUNC) were treated with investigated compounds as indicated. Following collection, the cells were fixed with ethanol. The fixed cells were washed with PBS and incubated with RNase A (1 mg/mL) for 30 min at 37 °C. Just prior to flow-cytometric analysis, the cells were stained with propidium iodide (PI) at a concentration of 400 μg/mL (Sigma-Aldrich, St. Louis, MO, USA). The cell cycle phase distribution was analyzed by an FACS Calibur Becton Dickinson flow cytometer using Cell Quest computer software (Becton Dickinson, Heidelberg, Germany).
4
Flow Cytometry Immunophenotyping Protocol
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Cell suspensions were stained in PBS with 5% FCSi, 0.05% sodium azide (staining buffer) and exhaustively washed before fluorescence-activated cell sorting (FACS). Fluorochrome-coupled antibodies against the surface antigens, CD3, CD4, CD8, CD11c, CD19, CD25, CD40, CD69, CD80, CD86, MHC-II, ICOS, NK1.1 and appropriate isotype controls were purchased from eBioscience (San Diego, CA, USA) or BioLegend (San Diego, CA, USA). Data were acquired on a FACSCalibur Becton Dickinson flow cytometer, or FACSCanto or BD LSRFortessa (BD Biosciences, San Jose, CA, USA) flow cytometers, and analyzed using FACSDiva (BD Biosciences) or FlowJo software (Tree Star, Ashland, OR, USA), version 10.0.
5
Telmisartan Induces Apoptosis Signaling
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After 24 h (for phospho-p38) or 48 h of treatment (for cleaved caspase 3) with 50 μM telmisartan, the cells were trypsinized, fixed, permeabilized, and then blocked with 1.5% BSA in PBS. Cells were then incubated with PE mouse anti-p38MAPK pT180/pY182 (612565, BD Biosciences, Franklin Lakes, USA) antibody or FITC rabbit anti-active caspase-3 antibody (51-68654x, BD Biosciences) for 1 h in the dark at room temperature. After washing, the cells were resuspended in PBS and analyzed using a FACS Calibur Becton Dickinson flow cytometer.
6
ROS Detection and Mitochondrial Potential Assay
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For the detection of ROS, 2 × 105 A375 or HTB140 cells were treated with 50 μM telmisartan, 100 μM pioglitazone, 100 μM losartan, or 50 μM telmisartan in the presence of 1 mM L-GSH, 10 mM NAC, or 100 μM apocynin for 24 h. The cells were stained with 50 μM H2DCFDA (D3999, Thermo Fischer Scientific, Schwerte, Germany) for 30 minutes at 37°C. For the measurement of the mitochondrial potential, A375 cells were treated with 50 μM telmisartan or 100 μM pioglitazone for 24 h, and then stained with 2.5 μg/mL of Rhodamine123 (R302, Thermo Fischer Scientific) in PBS for 1 h at 37°C, and analyzed using a FACS Calibur Becton Dickinson flow cytometer.
7
Apoptosis Evaluation of PEI/MNP/CIS Complexes
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Apoptosis was detected using annexin V-FITC/PI staining. The A2780/CP and A2780 cells were treated with 1 µg/mL PEI and 2.5 µg/mL CIS, and with PM/C and PMC complexes (at concentrations of 1 µg/mL PEI, 1 µg/mL MNPs, and 2.5 µg/mL CIS) in the presence and absence of SMF for 48 h, and were prepared in RPMI medium supplemented with 10% FBS in 6-well cell culture plates and incubated at 37°C and 5% CO2 in a humidified incubator. The cells were then collected and labeled with annexin V/PI in 1X binding buffer for 15 min. Apoptotic and necrotic cells were evaluated using a FACSCalibur Becton-Dickinson flow cytometer. The flow cytometric data were analyzed using FlowJo software. The total number of apoptotic and necrotic cells was defined as the sum of the Annexin V+/PI− and Annexin V+/PI+ populations; the cell populations in the four quadrants of the dot plot were analyzed as follows: the Q1 quadrant represented necrosis; Q2, late apoptosis; Q3, early apoptosis; and Q4 viable cells (47 (link), 48 ).
8
Cell Cycle Analysis by Flow Cytometry
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Briefly, 2 × 105 cells/Petri dish (dimensions 60 × 15 mm, NUNC) were treated with investigated drugs as indicated. After collection, cells were fixed with ethanol and stained with propidium iodide (PI) (Sigma-Aldrich, St. Louis, USA)141 . Cell cycle phase distribution was analyzed by FACS Calibur Becton Dickinson flow cytometer using Cell Quest computer software (Becton Dickinson, Heidelberg, Germany)
9
ROS Detection in Macrophages
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After exposure to the DAMs, RAW-264.7 macrophages were detached and cell suspensions were incubated with 100 μM of 2′,7′-dichlorofluorescein diacetate (DCFH/DA, Serva, Heidelberg, Germany) during 30 min at 37 °C. DCF fluorescence depends directly on the intracellular content of reactive oxygen species (ROS) and was measured in a FACScalibur Becton Dickinson flow cytometer with a 530/30 filter, exciting the sample at 488 nm. In each sample, 104 cells were analyzed.
10
Macrophage Size and Complexity Analysis
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After detachment of RAW 264.7 and J774A.1 macrophages, cell size and complexity were analyzed through the forward angle (FSC) and side angle (SSC) scatters, using a FACScalibur Becton Dickinson Flow Cytometer.
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