The distribution of cell cycle was detected using the cell cycle detection kit (Multi Sciences, Hangzhou, China) in accordance with the manufacturer’s protocols. Briefly, cells were incubated with DNA staining solution and permeabilization solution at room temperature for 30 min in the dark. The DNA content was detected by flow cytometry using a FACS Calibur system (Becton Dickinson, Franklin Lakes, NJ, USA), and the data were analyzed using FlowJo FACS analysis software (TreeStar, Ashland, OR, USA). The FITC-Annexin V apoptosis detection kit I (BD Pharmingen, San Diego, CA, USA) was used to detect apoptosis based on the manufacturer’s protocols. Cells were suspended in FITC-Annexin V binding buffer and then stained with FITC-Annexin V and PI for 15 min at room temperature in the dark. The stained cells were analyzed using a flow cytometer (Beckman Coulter, Miami, FL, USA).
Cell cycle detection kit
Manufactured by MultiSciences Biotech
Sourced in China
The cell cycle detection kit is a laboratory tool designed to analyze and monitor the different phases of the cell cycle. It provides a standardized method for the identification and quantification of cells in various stages of the cell cycle.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Cyclin d1, by Abcam (2 mentions)
Annexin 5 apc pi apoptosis detection kit, by MultiSciences Biotech (2 mentions)
Facscalibur system, by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Flow cytometry, by BD (1 mentions)
Recombinant mouse lefty 1, by R&D Systems (1 mentions)
α sma, by Abcam (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Cell counting kit 8 cck 8, by MultiSciences Biotech (1 mentions)
Cytochrome c, by Abcam (1 mentions)
Annexin 5 fitc pi apoptosis kit, by MultiSciences Biotech (1 mentions)
Pd0325901, by MedChemExpress (1 mentions)
Phospho pdk1 ser241, by Cell Signaling Technology (1 mentions)
Phospho mek1 2 ser217 221, by Cell Signaling Technology (1 mentions)
Bcl 2, by Abcam (1 mentions)
Apc annexin 5 and 7 aad apoptosis detection kit, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Apoptosis detection kit, by MultiSciences Biotech (1 mentions)
16 protocols using cell cycle detection kit
1
Cell Cycle and Apoptosis Analysis
2
Cell Cycle Analysis by Flow Cytometry
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Following 48 h from transfection, cells were fixed (using 75% ethanol at − 20 °C), resuspended in phosphate-buffered saline (PBS) and stain treated through Cell Cycle Detection Kit (MultiSciences, China), followed by flow cytometry analyses, with the results being assessed using FlowJo V10.
3
Cell Cycle Analysis by Flow Cytometry
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For cell-cycle analysis, cells underwent transfection for 48 hours (37°C, 5% CO2) and treatment with Cell cycle Detection Kit (MultiSciences, China) as directed by the manufacturer. In brief, after two phosphate-buffered saline (PBS) washes, the cells were treated with 10 μL permeabilization solution and stained with 1 mL DNA-staining solution at ambient, shielded from light, for 30 min. Finally, flow cytometry (BD Biosciences, USA) was carried out, and the Verity Software House software (VHS, USA) was used for analysis.
4
Cell Cycle Analysis via Flow Cytometry
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For the different phases of cell-cycle analysis, transfected cells were plated in a six-well transwell plates (5 × 104 cells/well). Cell-cycle progression was determined using a Cell Cycle Detection Kit (MultiSciences, China). After transfection for 48 h, the cells were washed with PBS and collected by centrifugation, and then treated with 10 μL permeabilization solution and 1mL DNA-staining solution. Finally, incubated for 30 min in the dark at room temperature and analyzed via flow cytometry (BD Biosciences, USA).
5
Metformin Effects on Cell Cycle
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The OSCC cells (2.5 × 105/well) in good condition were seeded in 6-well plates. Then, the cells were exposed to metformin (0, 12, 24 mM) for 48 h. We used a cell-cycle detection kit (MultiSciences, Hangzhou, China) to detect the cell-cycle distribution. According to the instructions, cells were collected followed by being washed with PBS. DNA Staining Solution (1 mL) was used to resuspend the cells and the permeabilization solution (10 μL) was added to cells. The cells were incubated in the dark. All samples were analyzed by flow cytometry.
6
Cell Cycle Analysis by Flow Cytometry
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A total of 2 × 105 cells was plated in 6‐well plates per well and cultured overnight. Then, all cells were synchronized in serum‐free medium for 48 hours. After that, the cells were resuspended in complete medium for another 24 hours. The cells in each group were fixed with 75% ethanol overnight and washed with 1× phosphate buffer saline (PBS) twice. According to the protocol of the cell cycle detection kit (Multi Sciences), the cells were resuspended with 400 μL of 1× binding buffer and stained with 20 μL of propidium iodide (PI) for 20 minutes at room temperature in the dark. The cell cycle was analyzed immediately with a flow cytometer (Becton‐Dickinso, FACSCalibur). The percentage of cells at each phase of the cell cycle was obtained by Cell Quest software (Becton‐Dickinso, FACSCalibur).
7
Molecular Regulation of Cellular Processes
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Dulbecco’s Modified Eagle’s Medium (DMEM; high glucose), trypsin/EDTA solution, and fetal bovine serum (FBS) were purchased from the Gibco® (Thermo Fisher Scientific, Waltham, MA, USA). Recombinant mouse Lefty-1 and TGF-β1 were obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Quantitative real-time polymerase chain reaction (PCR) kit was obtained from Hoffman-La Roche Ltd. (Basel, Switzerland), and Cell Counting Kit-8 (CCK)-8 and the cell cycle detection kit were acquired from MultiSciences Biotech Co., Ltd. (Hangzhou, Zhejiang, People’s Republic of China). Antibodies to fibronectin, collagen I(A1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), bone morphogenetic protein (BMP)-5, and proliferating cell nuclear antigen (PCNA) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). α-SMA and cyclin D1 were obtained from Abcam plc (Cambridge, UK). All other antibodies used in this study were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
8
Cell Cycle Analysis by Flow Cytometry
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Cell cycle progression was determined using a cell cycle detection kit (MULTI SCIENCES, Hangzhou, China). Briefly, 5 × 105 cells were harvested, washed twice with phosphate-buffered saline (PBS), collected by centrifugation at 300 × g for 5 min at room temperature, and treated with 10 μL permeabilization solution and 1 mL DNA staining solution. Finally, the cells were incubated for 30 min in the dark at room temperature and analyzed using a flow cytometer (BD Biosciences, San Jose, CA, USA).
9
Cell Cycle Progression Analysis
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Cell cycle progression was assessed using a Cell Cycle Detection Kit (MultiSciences, Hangzhou, China) following manufacturer’s description. In short, CRC cells in six-well plates were harvested, washed with PBS, treated with 10 μL of permeabilization solution and 1 mL of DNA staining solution for 30 min in the dark at RT and subjected to flow cytometry (BD Biosciences, San Jose, CA, USA). Cells at various cell cycle phases were assessed using FlowJo software (Version 10.6.2).
10
Apoptosis Regulation by Compound 9za
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Compound 9za (HPLC purity: 96.9%) was synthesized by Central South University. The Annexin V-FITC/PI Apoptosis Kit, Cell Cycle Detection Kit, Colorimetric TUNEL System, Mitochondrial Membrane Potential Assay Kit with JC-1 and MTT were purchased from MultiSciences (Hangzhou, China), KeyGEN (Nanjing, China), Promega (Madison, USA), Beyotime (Shanghai, China) and Sigma-Aldrich (St. Louis, Missouri, USA), respectively. Antibodies against active caspase-3, active caspase-9, Akt, phospho-Akt (Ser473), BAX, Bcl-2, CDK4, CDK6, Cyclin D1, Cytochrome C, PARP were purchased from Abcam (Cambridge, UK). Antibodies to detect the protein levels of ERK1/2, phospho-ERK1/2 (Thy202/Tyr204), MEK1/2, phospho-MEK1/2 (Ser217/221) and phospho-PDK1 (Ser241) were obtained from Cell Signaling Technology (Boston, USA). PD0325901 and BX517 were obtained from MedChemExpress (Shanghai, China).
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