Cell cycle and cell apoptosis analysis was carried out by flow cytometry. Cells (5×105/well) were seeded in 6-well cell culture plates and incubated in a humidified atmosphere of 5% CO2 at 37°C. Twenty-four hours later, cells were harvested with 0.5% trypsin and centrifuged. For cell cycle analysis, cells were fixed in ice-cold 70% ethanol overnight at 4°C. After being washed thrice with cold PBS, cells were resuspended in 500 µl of PBS, and 10 µl RNAseA was added for 5 min. Subsequently, 10 µl PI was added into the cell resuspension solution, and incubated for 30 min at 4°C. The cells were finally washed twice with PBS before analysis. Apoptotic analysis was performed using the Hoechst 33342/PI Apoptosis assay kit (BestBio, Shanghai, China). The samples were washed twice with ice-cold PBS and resuspended in 500 µl staining buffer, and then incubated with 5 µl of Hoechst 33342 and 5 µl of PI in the dark for 20 min at 4°C. The cells were finally washed twice with PBS before analysis. Analysis was performed on MoFlo™XDP High-Performance Cell Sorter (Beckman Coulter) and the data were analyzed with the summit v5.2 Software (Beckman Coulter).
Summit v5
Manufactured by Beckman Coulter
Sourced in United States
The Summit v5.2 software is a data analysis and reporting platform designed for use with Beckman Coulter laboratory equipment. It provides tools for data management, visualization, and report generation.
Automatically generated - may contain errors
Lab products found in correlation
Moflo xdp, by Beckman Coulter (7 mentions)
Moflo xdp high performance cell sorter, by Beckman Coulter (4 mentions)
Hoechst 33342 pi apoptosis assay kit, by BestBio (3 mentions)
Moflow xdp, by Beckman Coulter (2 mentions)
Accutase, by STEMCELL (2 mentions)
Cell cycle detection kit, by Keygen Biotech (2 mentions)
Cyan adp, by Beckman Coulter (2 mentions)
Cd45 pe, by BioLegend (2 mentions)
Moflo xdp70, by Beckman Coulter (2 mentions)
Gamunex, by Baxter (2 mentions)
Apc cd151, by BioLegend (2 mentions)
Percp cy5.5 cd3, by BioLegend (2 mentions)
Cd34 apc, by BioLegend (2 mentions)
Gallios 561, by Beckman Coulter (2 mentions)
Facs analysis, by Beckman Coulter (1 mentions)
Fura 2 am, by Beyotime (1 mentions)
Cell cycle staining kit, by Liankebio (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Hoechst 33342, by Keygen Biotech (1 mentions)
Stempro accutase, by Thermo Fisher Scientific (1 mentions)
22 protocols using summit v5
1
Cell Cycle and Apoptosis Analysis
2
Intracellular Calcium Concentration Analysis
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The fluorescent dye Fura-2 AM (Beyotime Institute of Biotechnology, Haimen, China) penetrates the cell membrane and binds calcium ions to produce strong fluorescence under 330–350 nm excitation light and weak fluorescence under 380 nm excitation light. The ratio of 340 and 380 nm fluorescence is routinely used to evaluate the intracellular calcium concentration. Fura-2 staining was performed according to the manufacturer’s instructions in the present study. Afterward, the cells were washed twice and resuspended in PBS for fluorescence-activated cell sorting (FACS) analysis (Beckman Coulter). The Ca2+ concentration data were analyzed using Summit v5.2 software (Beckman Coulter).
3
Measuring Intracellular ROS and Ca2+ Levels
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Cells were digested with 0.25% 1X Trypsin EDTA and cultured in RPMI-1640, and 5 µl 2,7-dichlorofluorescin diacetate (DCFH-DA) from a ROS kit (Applygen Technologies, Inc., Beijing, China) was added to the test tube. The blank group contained no reagents, whereas 5 µl DCFH-DA and 5 µl reactive oxygen donor was added to the positive control group and incubated in a constant temperature water bath at 37°C for 20 min. Subsequently, cells were washed with 1X PBS, and then resuspended in 500 µl 1X PBS. ROS was detected with a flow cytometer (Beckman Coulter, Inc.). For the Ca2+ concentration, following digestion of the cells with 0.25% 1X Tryspin EDTA, the cells were resuspended with the culture medium. A total of 1 µl Fluorescein-2 AM Ca2+ fluorescent probe from a Ca2+ analysis kit (S1056; Beyotime Institute of Biotechnology) was added to each tube of the sample to be tested. Subsequently, 1 µl dimethyl sulfoxide solution was added into the cell suspension of the control group, and then the tube was inverted and mixed thoroughly every 3-5 min to allow full contact between the cells and the Ca2+ fluorescence probe. The cells were then washed 2-3 times with 1X PBS and the cells were resuspended by adding 500 µl 1X PBS. The Ca2+ concentration was detected with a flow cytometer (Beckman Coulter, Inc.). The data were analyzed with Summit v5.2 Software (Beckman Coulter, Inc.).
4
Cell Cycle and Apoptosis Analysis by Flow Cytometry
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Prior to flow cytometric analyses, cells (5 × 105/well) were seeded in 6-well cell culture plates and cultured in a humidified atmosphere of 5% CO2 at 37°C for 24 h before harvesting with 0.5% trypsin and centrifugation. For cell cycle analysis, cells were fixed in ice-cold 70% ethanol overnight at 4°C. The fixed cells were then resuspended in 500 μl phosphate-buffered saline (PBS) to which 10 μl RNase A was added for a 5-min incubation followed by three washes with cold PBS. Next, 10 μl propidium iodide (PI) solution was added to the resuspended cell solution for incubation at 4°C for 30 min. Finally, the cells were washed twice with PBS before flow cytometric analysis of cell cycle distribution. For flow cytometric detection of apoptotic cells, the Hoechst 33342/PI Apoptosis Assay Kit (BestBio, Shanghai, China) was used. Briefly, the harvested cells were washed twice with ice-cold PBS and resuspended in 500 μl staining buffer. Then, the resuspended cell solution was incubated with 5 μl Hoechst 33342 and 5 μl PI solution in darkness for 20 min at 4°C. Finally, the cells were washed twice with PBS before flow cytometric analysis of cell apoptosis. All flow cytometric analyses were performed using a MoFloTM XDP High-Performance Cell Sorter (Beckman Coulter). The data for cell cycle distribution and apoptosis were analyzed using Summit v5.2 Software (Beckman Coulter).
5
Cell Cycle and Apoptosis Analysis
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Cells were harvested with 0.5% trypsin and centrifuged at 1,000 × g for 5 min at room temperature. Cell cycle analysis was performed using a cell cycle staining kit (LiankeBio; cat. no. CCS012), cells were fixed in ice-cold 70% ethanol overnight at 4°C. Following being washed thrice with cold PBS, cells were suspended in 500 µl PBS, and 10 µl RNase A was added for 5 min at room temperature. Subsequently, 10 µl propidium iodide (PI; LiankeBio; cat. no. CCS012) was added into the cell resuspension solution, and incubated for 30 min at 4°C. The cells were finally washed twice with PBS prior to analysis. As for apoptotic analysis, it was performed using a Hoechst 33342/PI Apoptosis Assay kit (Shanghai BestBio Biotechnology, Shanghai, China), according to the manufacturer's protocols. The samples were washed twice with ice-cold PBS and suspended in 500 µl staining buffer, and then incubated with 5 µl Hoechst 33342 and 5 µl PI in the dark for 20 min at 4°C. The cells were finally washed twice with PBS prior to analysis. Analysis was performed on a MoFlo™ XDP High-Performance Cell Sorter and the data were analyzed with Summit v5.2 Software (both from Beckman Coulter, Inc.).
6
Screening for Methanol-Utilizing Clones
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After about 10 h of cultivation, the libraries were transferred into fresh M9 medium with 2 g/L glucose and induced by adding 5 μg/L tetracycline and 1 μΜ IPTG for another 6 h of cultivation. Then 500 mM methanol was added as a substrate, and the culture was incubated for another 40 min. To avoid evaporation of methanol, the 96-well plates and shake flasks were covered with a sealing membrane. For screening positive clones, the culture was diluted by 20-fold using M9 medium and subjected to FACS analysis, which was performed using a Beckman Coulter MoFlo XDP. Green fluorescence was measured using the 488 nm laser and 525 nm filter. Cells were gated based on FSC-H and SSC-H, and 10,000 events falling into this window were recorded. The positive clones in the mutation libraries, which had relatively high fluorescence signals (about the top 0.01%), were collected onto LB plates, and then cultured at 37 °C for 12 h. All the data were analyzed using Beckman Summit v5.2 software.
7
Hoechst 33342 Dye-Based SP Cell Sorting
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The sorting and analysis of the SP cells was performed according to the methods described by Vieyra et al (32 (link)). In brief, the cells (1×106 cells/ml) were cultured in prepared DMEM, containing 2% FBS and 10 mmol/I HEPES. Subsequently, 5 µg/ml Hoechst 33342 dye was added to the cells in the presence or absence of 10 µmol/l FTC. Following incubation for 90 min, with intermittent shaking at 37°C, the cells were washed twice with ice-cold PBS. The Hoechst dye was excited at 355 nm, and the fluorescence profile was measured during the analysis (blue, 402–446 nm; red, 650–670 nm; MoFlo™ XDP; Beckman Coulter). The Summit v5.2 Software (Beckman Coulter) was used for the analysis.
8
Evaluating TPPU's Effect on TGF-β1-Induced Cell Cycle
9
Tucatinib's Effects on ABCG2 Expression
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The HL60/ABCG2 cell lines (1×105) were cultured in 6-well plates, containing various concentrations of tucatinib (0.1, 0.2 and 0.4 µM) for 24 h. Subsequently, 0.5 µg/ml Hoechst 33342 dye was added, followed by a further incubation for 30 min at 37°C. Finally, the cells were washed with ice-cold PBS, three times and re-suspended in PBS for flow cytometric analysis (MoFlo™ XDP; Beckman Coulter). The Summit v5.2 Software (Beckman Coulter) was used for the analysis.
10
Apoptosis Analysis by Flow Cytometry
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Cell apoptosis was analyzed by flow cytometric analysis using a MoFlo™ XDP High-Performance Cell Sorter (Beckman Coulter, Brea, CA, uSA), propidium iodide (PI) and Hoechst 33342 double staining (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). Briefly, the HeLa cells (HeLa, HeLa/vector and HeLa/CD24) were seeded at a density of 1x10 6 cells/well into 6-well culture plates. The cells were collected in an Eppendorf tube at 24 h and washed twice with PBS by centrifugation. The supernatants were discarded. To detect apoptosis, 500 µl PBS, 5 µl Hoechst 33342 and 5 µl PI were added to each tube, and the contents of the tube were mixed in the dark at room temperature for 15 min, followed by FCM testing (Beckman Coulter). Data were acquired and analyzed using Summit v5.2 software (Beckman Coulter).
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