The cells were stained with propidium iodide (PI) to conduct cell cycle analysis using a cell cycle staining kit (Beyotime Biotechnology, China) according to the manufacturer’s protocol and counted using a FACSCalibur flow cytometer.
Facscalibur flow cytometer
Manufactured by Beyotime
Sourced in China
The FACSCalibur flow cytometer is a compact and versatile instrument designed for multi-parameter analysis of cells and particles in suspension. It utilizes fluorescence-activated cell sorting (FACS) technology to detect and quantify various cellular properties, including size, granularity, and expression of specific markers. The FACSCalibur is capable of analyzing a wide range of sample types and provides reliable and reproducible data for research and clinical applications.
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Lab products found in correlation
Cell cycle staining kit, by Beyotime (1 mentions)
Annexin 5 pe apoptosis detection kit, by Beyotime (1 mentions)
C1052, by Beyotime (1 mentions)
Kga101, by Keygen Biotech (1 mentions)
Facscalibur flow cytometer, by Keygen Biotech (1 mentions)
Cell cycle and apoptosis analysis kit, by Beyotime (1 mentions)
Dcfh da, by Beyotime (1 mentions)
Cell counting kit 8 cck 8, by Beyotime (1 mentions)
Jc 1 staining solution, by Beyotime (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Annexin 5 fitc pi cell apoptosis detection kit, by Beyotime (1 mentions)
Facscalibur system, by BD (1 mentions)
8 protocols using facscalibur flow cytometer
1
Cell Cycle Analysis by PI Staining
2
Apoptosis Quantification by Flow Cytometry
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Flow cytometry experiments were utilized to detect the apoptosis rate of various groups. The PE Annexin V Apoptosis Detection Kit (Beyotime Biotechnology, Shang Hai, China, cat: 559763) was used for staining, and a FACSCalibur flow cytometer was used for counting.
3
Nano-SA-TCPP Cancer Therapy Evaluation
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Hela cells were also the probe cells to evaluate the cancer therapy performance of Nano-SA-TCPP. Specifically, Hela cells were incubated in the 25 cm2 cell-culture flask and then the cells (1 × 104 cells per well) were seeded into two 96-well plates by detaching from the flask. After seeding, the Hela cells were exposed to Nano-SA-TCPP of different concentrations for 24 h.
After that, each well was irradiated under different wavelengths for 10 min, and during the irradiating other wells were kept in the dark with tinfoil. The light source was a xenon lamp source, PLS-SXE 300D, Beijing Perfectlight Technology Co., Ltd. with band-pass filters (600 ± 15, 650 ± 15, 700 ± 15 nm), and the irradiance was 0.1 W cm−2. For the cell viability after irradiation, a mixture of CCK-8 and DMEM (1 : 10) was added to the 96-plate. The cell viability was calculated as the ratio of the absorbance of the wells. The absorbance at 450 nm was measured by Thermo Multiskan FC. The cell flow cytometry analysis was conducted on a BD Biosciences FACSCalibur flow cytometer with the ROS probe of DCFH-DA purchased from Beyotime Biotechnology. The data were analyzed by FlowJo Software.
After that, each well was irradiated under different wavelengths for 10 min, and during the irradiating other wells were kept in the dark with tinfoil. The light source was a xenon lamp source, PLS-SXE 300D, Beijing Perfectlight Technology Co., Ltd. with band-pass filters (600 ± 15, 650 ± 15, 700 ± 15 nm), and the irradiance was 0.1 W cm−2. For the cell viability after irradiation, a mixture of CCK-8 and DMEM (1 : 10) was added to the 96-plate. The cell viability was calculated as the ratio of the absorbance of the wells. The absorbance at 450 nm was measured by Thermo Multiskan FC. The cell flow cytometry analysis was conducted on a BD Biosciences FACSCalibur flow cytometer with the ROS probe of DCFH-DA purchased from Beyotime Biotechnology. The data were analyzed by FlowJo Software.
4
Apoptosis and Cell Cycle Analysis in A549 and H1299 Cells
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After transfection for 48 h, the A549 and H1299 cells were harvested, washed twice with ice-cold PBS, and resuspended in 1× binding buffer. For cell apoptosis analysis, cells were incubated in Annexin-V/PI double staining solution (KGA101, KeyGen Biotech, Nanjing, China) for 20 min, according to the manufacturer's instructions, and then analysed using a FACSCalibur flow cytometer. The cell cycle was evaluated using a cell cycle and apoptosis analysis kit (C1052, Beyotime) according to the manufacturer's instructions using a FACS Calibur flow cytometer.
5
Measuring Cellular Reactive Oxygen Levels
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Cellular ROS levels were detected as described previously (Chen et al., 2018 ). Briefly, cells were treated with the indicated chemical agents for 2 h. Then the cells were stained with 10 μM DCFH-DA (Beyotime, Shanghai, China) for 30 min at 37°C and analyzed by the FACSCalibur flow cytometer.
6
Inducing Senescence in HUVECs
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H2O2 was used to stimulate and induce cell senescence in HUVECs. The cells were randomly divided into 5 groups and treated with different concentrations of H2O2 (0, 10, 50, 100 and 500 µM). The blank group (0 µM H2O2) was cultured in normal medium, whereas the H2O2 group was incubated with normal medium at different concentrations of H2O2 for 24 h. The cell viability, proportion of senescent cells and cell cycle in each group were determined by Cell-Counting-Kit 8 (CCK-8; Beyotime Institute of Biotechnology), SA-β-galactosidase (SA-β-gal; Beyotime Institute of Biotechnology) and FACSCalibur Flow Cytometer (cat. no. 342973; BD Biosciences; Becton, Dickinson and Company).
7
Evaluating Apoptosis and Mitochondrial Dynamics in Osteoblasts Infected with E. faecalis
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To evaluate the apoptotic rate of cells (in early and late phases), primary osteoblasts were incubated with E. faecalis OG1RF at MOIs of 10, 100, 500, and 1,000 for 6 and 12 h, respectively. The infected cells were incubated with staining solution containing propidium iodide (PI) and FITC Annexin V (#556,570; BD Biosciences, San Diego, CA, USA) at 25 °C for 15 min. The apoptotic cells in early (FITC Annexin V+/PI−) and late (FITC Annexin V+/PI+) phases were analysed using a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA, USA).
To detect the change in mitochondrial membrane potential (ΔΨm), primary osteoblasts were incubated with E. faecalis OG1RF at MOIs of 10, 100, 500, and 1,000 for 6 and 12 h, respectively. The infected cells were incubated with 0.5 mL of JC-1 staining solution (#C2006; Beyotime, Shanghai, China) and evaluated using a FACSCalibur flow cytometer. The ΔΨm was estimated by calculating the ratio of red/green fluorescence intensities as previously reported [12 ].
To detect the change in mitochondrial membrane potential (ΔΨm), primary osteoblasts were incubated with E. faecalis OG1RF at MOIs of 10, 100, 500, and 1,000 for 6 and 12 h, respectively. The infected cells were incubated with 0.5 mL of JC-1 staining solution (#C2006; Beyotime, Shanghai, China) and evaluated using a FACSCalibur flow cytometer. The ΔΨm was estimated by calculating the ratio of red/green fluorescence intensities as previously reported [12 ].
8
Cell Cycle and Apoptosis Analysis
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The DNA contents of the cells were determined by flow cytometry to estimate the percentages of cells in different phases of the cell cycle. The treated cells were collected, washed twice with cold PBS, and fixed in 70% ethanol at −20 °C overnight. The cells were resuspended in PBS containing 50 mg/ml propidium iodide (PI) and 10 mg/ml DNase-free RNase immediately before flow cytometry. A FACSCalibur system (Becton Dickinson, San Diego, CA, USA) equipped with CellQuest software was used for flow cytometry analysis.
For apoptosis analysis, PASMCs were seeded at 3 × 103 cells/well in 96-well plates using triplicate wells for each transfection. After 48 h, the cells treated under various conditions were incubated in 1.5% H2O2 for 24 h. The cells were then starved in medium containing 0.1% FBS; stained with Annexin V-FITC and PI reagents in binding buffer using the Annexin V-FITC/PI cell apoptosis detection kit (Beyotime Institute of Biotechnology, Beijing, China); and analyzed using a FACSCalibur flow cytometer.
For apoptosis analysis, PASMCs were seeded at 3
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