The cells were seeded and grown in 60-mm culture plates and treated with increasing concentrations of CA or DMSO for 48 h, respectively. The cells were then trypsinized, collected, and washed using phosphate-buffered saline (FBS). After resuspending the cells, pre-cooled absolute ethanol was added to fix cells overnight. According to the manufacturer’s instruction of Cell Cycle and Apoptosis Analysis Kit (Beyotime, Jiangsu, China), 0.5 mL staining buffer, 25 µL propidium iodide staining solution (20X), and 10 µL RNase A (50X) were added to each sample, and the cell cycle distribution (5,000 events) was analyzed using flow cytometry (Beckman Coulter, United States) after incubation for 30 min.
Flow cytometry
Manufactured by Beyotime
Sourced in China, United States
Flow cytometry is an analytical technique used to measure and analyze the physical and chemical characteristics of cells or particles suspended in a fluid stream. It allows for the rapid measurement and analysis of multiple parameters of individual cells or particles passing through a laser beam.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Beyotime (2 mentions)
Annexin 5 fitc, by Beyotime (2 mentions)
Cell cycle and apoptosis analysis kit, by Beyotime (1 mentions)
Luciferase based enhanced atp assay kit, by Beyotime (1 mentions)
Multi function microplate reader, by PerkinElmer (1 mentions)
Gsh assay kit, by Cayman Chemical (1 mentions)
Bodipy c 11 dye, by Beyotime (1 mentions)
Mda detection kit, by Beyotime (1 mentions)
Iron assay kit, by Beyotime (1 mentions)
Microplate reader, by PerkinElmer (1 mentions)
Fitc conjugated annexin 5, by Beyotime (1 mentions)
Jc 1 staining buffer, by Beyotime (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Propidium iodide staining solution, by Beyotime (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc and pi, by Beyotime (1 mentions)
Jc 1 dyeing working solution, by Beyotime (1 mentions)
12 protocols using flow cytometry
1
Cell Cycle Analysis by Flow Cytometry
2
Mitochondrial Membrane Potential and ATP Assay
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A JC-1 MMP measurement kit (Beyotime, Shanghai, China) was used to measure MMP (ΔΨm). Briefly, the indicated cells (2×105) were resuspended in 0.5 mL of JC-1 working solution and incubated for 20 min. The cells were washed and analyzed by flow cytometry (Biosciences, Franklin Lakes, NJ).
A luciferase-based enhanced ATP assay kit (Beyotime, Shanghai, China) was used to determine ATP levels. Briefly, the indicated cells were lysed and centrifuged at 12,000× g for 5 min. The supernatant was added to a 96-well plate containing ATP detection working solution. Luminescence was detected by a multifunction microplate reader (Perkin Elmer, USA). The protein concentration of each group and an ATP standard curve were used to calibrate the ATP levels in the cells.
A luciferase-based enhanced ATP assay kit (Beyotime, Shanghai, China) was used to determine ATP levels. Briefly, the indicated cells were lysed and centrifuged at 12,000× g for 5 min. The supernatant was added to a 96-well plate containing ATP detection working solution. Luminescence was detected by a multifunction microplate reader (Perkin Elmer, USA). The protein concentration of each group and an ATP standard curve were used to calibrate the ATP levels in the cells.
3
Ferroptosis Induction and Measurement
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SGC7901 and BGC823 cells were cotreated with erastin (5 mmol/L) or ferrostatin (1 mmol/L). After 48 h, the cell viability was analyzed by MTT assay. Elevated iron level and accumulated lipid reactive oxygen species (ROS) were representative characteristics of ferroptosis. We used an iron assay kit (Beyotime, China) to examine the level of intracellular Fe2+. The cells were homogenized to collect the supernatant, incubated with iron reducer, followed by labeling with iron probe. OD 590 nm was detected in a microplate reader (PerkinElmer, Waltham, MA, United States). For detection of lipid ROS, cells were stained with BODIPY C-11 dye (Beyotime) for 30 min, and subsequently detected by flow cytometry (BD Biosciences, Franklin Lakes, NJ, United States). The levels of malondialdehyde (MDA) and glutathione peroxidase (GSH) was measured by the MDA detection kit (Beyotime) and GSH assay kit (Cayman, Ann Arbor, MI, United States), respectively.
4
Apoptosis Measurement by Annexin V-FITC
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Cells were cultured overnight in 6-well plates and treated for 24 h with BSE. Apoptosis in the cultures was assessed using a kit based on staining with FITC-conjugated annexin V (catalog no. C1062S, Beyotime, Shanghai, China) and flow cytometry (Beckman Coulter, Brea, CA, USA). Cells were suspended in a mixture of 1X annexin V-FITC binding buffer (195 µL) and annexin V-FITC (5 µL), incubated at room temperature for 10 min, and centrifuged at 1,000 ×g for 5 min. The pellet was resuspended in binding buffer (190 µL), and propidium iodide (PI) working solution (10 µL) was added before the samples were analyzed using flow cytometry.
5
Cell Cycle and Apoptosis Analysis
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Flow cytometric analysis was performed to study cell cycle distribution and apoptosis level. Briefly, to carry out the cell cycle and apoptosis assays, treated cells were harvested and then washed twice with pre-cooled PBS. For cell cycle analysis, cells were fixed with 75% ice-cold ethanol at 4 °C overnight, followed by incubation with propidium iodide and RNase A for 15 mins at 37 °C. After washing with cold PBS three times, FACS caliber flow cytometry (Beckman Cytoflex) was used to acquire the DNA contents of labeled cells. For apoptosis analysis, Annexin V-fluorescein isothiocyanate (FITC)/PI straining was performed and sample acquisition was done using flow cytometry according to the manufacturer’s instructions (Beyotime, Shanghai, China). Collected data was analyzed with Flow Jo software.
6
Cell Viability Analysis of PC3/Doc Cells
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After treatment of PC3/Doc cells with chemicals for 1 and 5 days, cells were detected using propidium iodide (PI, Beyotime, Shanghai, China) staining with flow cytometry (BD Biosciences, San Jose, CA, USA).
7
Evaluating Cellular Stress Response Markers
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To evaluate ROS levels, kidney tissues were rinsed with PBS, resuspended in erythrocyte lysate, neutralized with an equal volume of PBS, centrifuged, and resuspended in PBS. ROS levels were detected using flow cytometry according to the manufacturer’s instructions (Beyotime, Shanghai, China).
To evaluate apoptosis, 1 × 106 cells were centrifuged and resuspended in 200 μL of PBS. 10 μL Annexin V-FITC and 10 μL PI (BD, New Jersey, USA) were gently mixed and incubated at 4 °C for 30 min in the dark. After the addition of 300 μL of PBS, the cells were subjected to flow cytometry.
To evaluate the mitochondrial membrane potential, 1 × 106 cells were resuspended in cell culture medium (0.5 mL) and incubated with JC-1 staining solution (0.5 mL) for 20 min. The cells were then centrifuged and resuspended in JC-1 staining buffer (Beyotime, Shanghai, China). Mitochondrial membrane potential was detected by flow cytometry.
To evaluate apoptosis, 1 × 106 cells were centrifuged and resuspended in 200 μL of PBS. 10 μL Annexin V-FITC and 10 μL PI (BD, New Jersey, USA) were gently mixed and incubated at 4 °C for 30 min in the dark. After the addition of 300 μL of PBS, the cells were subjected to flow cytometry.
To evaluate the mitochondrial membrane potential, 1 × 106 cells were resuspended in cell culture medium (0.5 mL) and incubated with JC-1 staining solution (0.5 mL) for 20 min. The cells were then centrifuged and resuspended in JC-1 staining buffer (Beyotime, Shanghai, China). Mitochondrial membrane potential was detected by flow cytometry.
8
Apoptosis Assay by Flow Cytometry
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Cells were seeded at a density of 2×105 cells per well in a 6-well plate and cultivated overnight. The cells were then treated with the control or drug-containing media for 72 h. Subsequently, the cells were collected and stained with Annexin V-FITC and propidium iodide (Beyotime Biotechnology, China) for 20 min and then analysed using flow cytometry (Beckman Coulter, USA). At least 5×104 cells were analysed for each sample.
9
Annexin V-FITC Apoptosis Assay
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Mouse GC1-spg cells were prepared by trypsin (Gibco) digestion and centrifuged (L550, Xiangyi, Changsha, China) at 1000g for 5 min. Then, the cells were resuspended in PBS and counted. Aliquots of 100 000 resuspended cells were centrifuged at 1000g for 5 min. The supernatant was discarded and 195 μl Annexin V-FITC (Beyotime, Shanghai, China) was added. Cells were resuspended and treated with 5 μl of Annexin V-FITC, 10 μl of propidium iodide staining solution (Beyotime) was added, and cells were subjected to flow cytometry (Biosciences, Franklin Lakes, NJ, USA).
10
Annexin V-FITC and PI Staining
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After ZnO NPs treatment for 24 hours, both floating and attached cells were harvested. Cells were stained with 5 μl of annexin V-FITC and PI (Beyotime, China) for 5 minutes in the dark and analyzed by flow cytometry (Beckman Coulter, United States).
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