Intracellular ROS generation was determined using peroxidesensitive fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFHDA, Molecular Probes). The cells were cultured into 6-well plates followed by exposure to the indicated experimental conditions, and the cells were incubated with 5 mM DCFH-DA for 20 mins at 37°C in the dark. Subsequently, the cells were rinsed with serum-free EGM-2 twice, and the representative images of ROS generation were captured using a Nikon Eclipse Ti-U epifluorescence microscope or a flow cytometry (FACS Calibur, Bio-Rad Laboratories, Inc., USA).
Facscalibur
Manufactured by Bio-Rad
Sourced in United States
The FACSCalibur is a flow cytometry instrument designed for multiparametric analysis of cells and particles. It utilizes two lasers to simultaneously detect and measure multiple fluorescent and light-scattering signals from individual cells or particles.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 fitc kit, by Beyotime (2 mentions)
Dcfh da, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fluorescein isothiocyanate fitc apoptosis kit, by Beyotime (1 mentions)
Cell cycle assay kit, by Abcam (1 mentions)
Modfit lt 3, by BD (1 mentions)
Ab11400, by Abcam (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Annexin 5 fitc pi apoptosis kit, by BD (1 mentions)
Annexin 5 fitc apoptosis kit, by Keygen Biotech (1 mentions)
Apoptosis assay kit, by Merck Group (1 mentions)
Cellquest software, by BD (1 mentions)
Quantibrite kit, by BD (1 mentions)
Matched isotype pe control, by Bio-Rad (1 mentions)
Verikine human ifn alpha elisa kit, by PBL Assay Science (1 mentions)
Recombinant b18r protein, by Thermo Fisher Scientific (1 mentions)
16 protocols using facscalibur
1
Intracellular ROS Generation Assay
2
Annexin V-FITC Apoptosis Assay in H9C2 Cells
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After treatment, H9C2 cells were stained with Annexin V‐fluorescein isothiocyanate (FITC) Apoptosis Kit (Beyotime, Jiangsu, China) in accordance with the manufacturer's instructions. Briefly, cells were harvested and washed three times with PBS. Subsequently, cells were resuspended in Annexin V binding buffer and stained with Annexin V‐FITC and propidium iodide in the dark for 15 minutes at room temperature. Stained cells were analysed by flow cytometry (FACS Calibur, Bio‐Rad Laboratories, Inc, USA) within 1 hour.
3
Cell Cycle Analysis by Flow Cytometry
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The procedures of cell cycle analysis were carried out following the manufacturer’s instructions of Cell Cycle Assay Kit (ab112116, Abcam). Attached cells were harvested cells and fixed in 70% ice-cold ethanol overnight at 4 °C. Finally, cells were stained with RNase A (10 mg/mL) and Propidium Iodide (50 mg/mL) before analyzed by a flow cytometer (FACSCalibur, Bio-Rad, Hercules, CA, USA). Data were analyzed using CELL Quest 3.0 software.
4
Cell Cycle Analysis of CME Treatment
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Cells (1 × 106 cells/well) were treated with various concentrations of CME (15, 25, and 40 μg/mL) for 24, 48, and 72 h. Cells were then collected and washed twice with 1 mL of ice-cold PBS, suspended in 1 mL of ice-cold 70 % ethanol (containing 1 × 105 cells) and kept overnight at 4 °C. The next day, cells were washed with PBS and incubated with 500 μL of PI staining solution (containing PI 40 μg/mL, RNase A 100 μg/mL and PBS) for 30 min in the dark at 37 °C. Then, samples were filtered using 70-μm filters into test tubes and detected by flow cytometry (FACSCalibur, Bio-Rad). Analysis of cell cycle status was performed using ModFit LT 3.0™ software (BD Biosciences).
5
Apoptosis Analysis of CENPF, CDK1 Knockdown
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Human SW13 cells transfected with siCENPF, CDK1, or siNC for 48 h were disposed with Annexin V-FITC kit (Beyotime Biotechnology, China) and analyzed by flow cytometry (FACSCalibur, Bio-Rad, USA) to detect cell apoptosis. Data were analyzed using FlowJo7.6 software.
6
Apoptosis Detection in Pancreatic Cancer Cells
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Tumor cell apoptosis was detected using flow cytometry using an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (cat. no. MR-KA020) from MingRui Biotechnology Co. Ltd., according to the manufacturer's protocol. Briefly, near-80% confluent PANC-1 and BXPC-3 cells were harvested using 0.25% trypsin (MingRui Biotechnology Co., Ltd.), washed twice in PBS and resuspended at a density of 1×106 viable cells/ml in Annexin V binding buffer (MingRui Biotechnology Co., Ltd.). Subsequently, 5 ml Annexin V/FITC, at a final concentration of 50 µg/ml, was added to the cell suspension with 10 µl propidium iodide, at a final concentration of 1 µg/ml. The cell solution was incubated for 15 min at room temperature in the dark. Following incubation, the stained cells were immediately analyzed using a flow cytometer (FACSCalibur; Bio-Rad Laboratories, Inc.). Each experiment was performed in triplicate and repeated at least three times.
7
Apoptosis Evaluation in SW13 Cells
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Human SW13 cells were transfected with siESM1 or control siRNA for 48 h. Cells were then disposed with Annexin V-FITC kit (Beyotime Biotechnology, China) and analyzed with flow cytometry (FACSCalibur, Bio-Rad, Hercules, CA, USA) to detect cell apoptosis. Data were analyzed using FlowJo7.6 software.
8
Apoptosis Assay for Neuronal Cells
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Neurons were digested with 0.125% trypsin for approximately 4 minutes. Cells were collected and placed in centrifuge tubes and made into a single-cell suspension. Thereafter, 1 mL of cells at 1 × 106/mL was centrifuged at 1,000 r/min for 10 minutes. The supernatant was discarded, and the cells were resuspended in 1 mL of cold PBS. Cells were resuspended in 200 μL binding buffer, and 10 μL annexin V-FITC and 5 μL propidium iodide were added in the dark at room temperature for 20 minutes. For the control, 400 μL of PBS was added instead of dye. Samples were filtered through a 200-mesh sieve and detected immediately with a flow cytometer (FACSCalibur; Bio-Rad) (Yang et al., 2009).
9
EGFR Expression in Glioblastoma Cell Lines
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Tumor cells (U87, U373, GBM2, GBM3, and GBM4) were disaggregated with Accumax (15 min, room temperature), and then they were stained with an antibody against EGFR conjugated with FITC (Abcam, Cambridge, UK, #ab11400) diluted in PBS-1% BSA (Staining buffer) for 30 min on ice. Cells were washed in PBS, treated with propidium iodide (5 μg/mL, Sigma-Aldrich) and analyzed by flow cytometry (FACSCalibur, Bio-Rad Laboratories, Hercules, CA, USA), using the FlowJo software (https://www.flowjo.com ).
10
Quantification of Cell Apoptosis
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Cell apoptosis was evaluated by flow cytometry using an Annexin V-FITC/PI apoptosis kit (BD Biosciences) according to the manufacturer's protocol. After cell harvesting, 100 µl binding buffer was added to prepare a cell suspension (1x106 cells/ml). Subsequently, cells were stained with 5 µl FITC-Annexin V and 5 µl PI at room temperature for 15 min in the dark. Cells were then resuspended in 400 µl binding buffer and analyzed by flow cytometry (FACSCalibur; Bio-Rad Laboratories, Inc.). Cells in early and late apoptosis was quantified using FlowJo 7.6.1 software (FlowJo LLC).
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