The immune phenotypes of cells were determined by flow cytometry to monitor the level of HSC differentiation and the presence of various cell types. Mononuclear cells (1 x 105 cells) from each culture condition were stained with the fluorescent dye-labeled monoclonal antibodies and incubated for 30 minutes at 4 °C in the dark. The cells were then washed twice with PBS by centrifugation at 800 g, 5 min at 4 °C and fixed with 1 % paraformaldehyde in PBS. The stained cells were analyzed by FACScan flow cytometry (Beckman Coulter, USA) to determine the expression pattern of lineage-associated antigens that indicate the various committed cellular subsets using a commercially available kit with fluorochrome-labelled monoclonal antibodies [(Biolegend): CD34+HLA-DR-, CD3+CD4+, CD3+CD8+, CD19+, CD14+, CD56+CD3-, CD56+CD3+, CD40+HLA-DR+].
Facscan flow cytometry
Manufactured by Beckman Coulter
Sourced in United States
The FACScan flow cytometry system is a compact and versatile instrument designed for multiparametric analysis of individual cells. It utilizes a laser-based detection system to measure various optical and fluorescent characteristics of cells, enabling researchers to identify and quantify diverse cell populations within a sample. The FACScan provides essential tools for a wide range of applications in fields such as immunology, cell biology, and disease research.
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Lab products found in correlation
Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit, by Vazyme (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Abcam (1 mentions)
Cell quest acquisition software, by BD (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by BD (1 mentions)
Cell quest software, by Beckman Coulter (1 mentions)
2 7 dichlorodihydrofluorescein diacetate h2dcfda, by Thermo Fisher Scientific (1 mentions)
Six well plate, by Corning (1 mentions)
10 protocols using facscan flow cytometry
1
Immune Phenotyping of Cell Differentiation
2
Apoptosis Assessment of Glioma Cells
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The Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (Vazyme) was utilized to assess the apoptosis rate of glioma cells according to the manufacturers’ instructions. Briefly, transfected T98G and LN229 cells were collected, resuspended, washed and then mixed with 5 µL Annexin V-FITC and 5 µL propidium iodide (PI); 15 min later, the apoptotic cells were determined with a FACScan® flow cytometry (Beckman Coulter, Atlanta, GA, USA).
3
Apoptosis Assay of HepG2 Cells
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The apoptosis of cells induced by the extract was assayed by treatment with Annexin V and propidium iodide (PI) double labeling according to the manufacturer’s instruction (BioVision Annexin V-FITC Apoptosis Detection Kit). HepG2 cells (2 × 106 cells/mL) were incubated in a 6 cm dish for 72 h in the presence or absence of the test samples. Cells were centrifuged to remove the medium, washed with PBS, and stained with Annexin V and PI in binding buffer. The stained cells were determined using FACScan flow cytometry (, Beckman Coulter, Indianapolis, IN, USA).
4
Cell Cycle Analysis of VEGF-Treated ASM Cells
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ASM cells were cultured in the complete medium with 1,25-(OH)2D3 for 2 h before treated or not with 50 ng/ml of VEGF for 48 h. All the cells were collected, and 1 × 106 cells were centrifuged, resuspended in ice-cold 70% ethanol and stored at −20 °C until further analysis. Washed cells were stained by 0.1% Triton X-100 in 0.01 M phosphate-buffered saline (pH 7.2) with 50 μg/ml propidium iodide (Sigma-Aldrich) and 1 mg/ml RNase A (Invitrogen), and incubated at 37 °C for 30 min in the dark. Samples of the cells were then analyzed for their DNA content using FACScan flow cytometry (Beckman, Miami, FL), and cell cycle phase distributions were analyzed by the Cell Quest acquisition software (BD Biosciences, Franklin Lanes, NJ). All experiments were performed in duplicate and repeated twice.
5
Cardiomyocyte Apoptosis Quantification
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The apoptosis rate of cardiomyocytes was determined via an Annexin V-FITC Apoptosis Detection Kit and a flow cytometer. Cardiomyocytes were cultivated in six-well culture plates with Dulbecco's modified Eagle medium with 10% FBS for 48 h. The medium was replaced with serum-free medium and cultivated for 24 h. Then, cells were pretreated with DA (1, 3, and 10 μmol/L) and captopril (5 μmol/L) for 35 min, followed by 0.1 μmol/L angiotensin II (Ang II) for another 48 h. After incubation, cells were cleaned with PBS and resuspended. Then, fluorescein-conjugated annexin V (5 mL) and propidium iodide reagent (5 mL) were added to cell suspensions. The mixture above was incubated for 15 min protected from light. Finally, the rate of apoptosis was determined and quantified by FACScan flow cytometry (Beckman Coulter).
6
Annexin V-FITC/PI Apoptosis Assay
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For analysis of cell apoptosis, an Annexin V-FITC/PI apoptosis detection kit was used (BD Bioscience, San Jose, CA, USA). According to the manufacturer’s instructions, cells were collected and washed with binding buffer and then were incubated for 15 min with 5 μL of annexin V-FITC and 5 μL of PI. The apoptosis rate of the cells was examined by FAC Scan flow cytometry from Beckman Coulter (Brea, CA, USA).
7
Annexin V-FITC and PI Staining
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After cells had been subjected to the treatments as described above, cells were washed twice gently with cold phosphate buffered saline (PBS). The apoptosis ratios of H9c2 cells were conducted using fluorescein isothiocynate (FITC)-conjugated Annexin V and propidium iodide (PI) staining method (Invitrogen, Carlsbad, CA, USA) on a FACScan flow cytometry (Beckman Coulter, Atlanta, GA, USA). The data were analyzed by using FlowJo software (TreeStar, Ashland, OR, USA).
8
Quantifying Apoptosis by Flow Cytometry
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Flow cytometry was used to quantitatively detect the apoptosis rate. After being treated with n-butyl-β-D-fructofuranoside at various concentrations (0, 50, and 75 μg/mL) for 48 h, cells were washed with PBS (pH 7.4) and fixed in 70% ethanol at 4°C overnight. After fixation, the cells were stained with PI at 1 mg/mL for 15 min at room temperature, determined by FACScan flow cytometry (Beckman, USA) and analyzed by CellQuest software (Beckman, USA).
9
Measuring Intracellular ROS and Mitochondrial Membrane Potential
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The method of ROS detection was described in our previous study 25 (link). 2',7'-Dichlorodihydrofluorescein diacetate (H2DCF-DA) (Molecular Probes, Eugene, OR, USA) was used to measure intercellular ROS production. The ΔΨm is defined as a change in the electrochemical gradient. The mitochondrial ΔΨm was measured using a fluorescent cationic dye, tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (Molecular Probes). JC-1 dye exhibits potential-dependent accumulation in mitochondria and is detected by a fluorescence emission shift from green (~529 nm) to red (~590 nm). The mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. HA22T/VGH cells were seeded in six-well plates (Corning, Inc., Corning, NY, USA) and incubated overnight, then underwent treatment for 24 h before being incubated with 2 μM JC-1 dye for 15 min at 37°C. The cells were subsequently trypsinized and resuspended for analysis by FACScan flow cytometry (Beckman Coulter-Epics XL) 26 (link). Data were analyzed using WinMDI 2.8 software (Scripps Research Institute, La Jolla, CA, USA), and a minimum of 1×104 cells per sample were evaluated.
10
Apoptosis Analysis via PI and FITC-Annexin V
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For cell apoptosis analysis, it was examined via PI and fluorescein isothiocynate (FITC)-conjugated Annexin V staining [20 ]. Briefly, cells were washed with PBS and fixed with 70% ethanol. Fixed cells were then washed twice with PBS and stained in PI/FITC Annexin V, and then incubated at room temperature in the dark for 1 h. FACScan flow cytometry (Beckman Coulter, USA) was used to analyze apoptotic cells and necrotic cells. The data were analyzed using FlowJo software.
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