For quantification of cell cycle stage distribution, cells were seeded in a 6-well plate at a density of 3 × 105 cells/well. After 24 h, cells were harvested and analyzed using propidium Iodide (PI) staining solution (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Subsequently the samples were analyzed using a BD FACSCanto II instrument (BD Biosciences, Franklin Lakes, NJ, USA). A total of 10,000 events were acquired for each sample. Data were analyzed with FACSDiva software (BD Biosciences).
Pi staining solution
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
PI staining solution is a laboratory reagent used for DNA content analysis. It binds to DNA and emits fluorescence upon excitation, allowing for the quantification of DNA content in cells.
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Lab products found in correlation
Accuri c6 flow cytometer, by BD (2 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Accouri c6 flow cytometer, by BD (1 mentions)
Facs flow cytometer, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Rnaase, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Collagenase 1, by Thermo Fisher Scientific (1 mentions)
Anti cd16 32 clone 93, by Thermo Fisher Scientific (1 mentions)
Binding buffer, by BD (1 mentions)
Verteporfin, by Merck Group (1 mentions)
Cyan adp analyzer, by Beckman Coulter (1 mentions)
Annexin 5 apoptosis detection kit apc, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by BD (1 mentions)
Acurri c6, by BD (1 mentions)
15 protocols using pi staining solution
1
Cell Cycle Analysis by Flow Cytometry
2
Cell Cycle Analysis by Flow Cytometry
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Cells were harvested in PBS and fixed by addition of ice-cold 70% ethanol with a Pasteur pipette during vortexing, as previously described [8 (link)]. Then, the cells were centrifuged at approximately 2000 rpm for 5 min and washed twice with PBS. Finally, the cell were stained by PI staining solution (Invitrogen) and analyzed by flow cytometry collecting 25,000 events per sample. The results were analyzed with FlowJo software.
3
Cell Fixation and Nuclear Protein Extraction
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Cells were harvested in PBS and xed by addition of ice-cold 70% ethanol with a Pasteur pipette during vortexing, as previously described [8] . Then, the cells were centrifuged at approximately 2,000 rpm for 5 minutes and washed twice with PBS. Finally, the cell were stained by PI staining solution (Invitrogen) and analyzed by ow cytometry collecting 25,000 events per sample. The results were analyzed with FlowJo software Nuclear Protein Extraction According to the methods described in a previous study [23] , cells were washed three times with precooled phosphate buffered saline (PBS) and subsequently scraped off with a cell scraper and collected in a 1.5 mL centrifuge tube. The cells were centrifuged at 1,000 rcf, 4°C, for 3 min. A protease inhibitor, cell lysis buffer, and cell membrane rupture solution were added into the pellet to lyse the cells on ice for 1 h, followed by 1,000 rcf centrifugation at 4°C for 20 min. The supernatant was discarded, and the pellet was then lysed with nuclear extraction lysis buffer on ice for 1 h, with vortexing every 5 h for complete lysis, which was followed by 12,000 rcf centrifugation at 4°C for 15 min to collect the supernatant (i.e., nuclear protein extract).
4
Cell Cycle Analysis by Flow Cytometry
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Cells were harvested in PBS and xed by addition of ice-cold 70% ethanol with a Pasteur pipette during vortexing, as previously described [8] . Then, the cells were centrifuged at approximately 2,000 rpm for 5 minutes and washed twice with PBS. Finally, the cell were stained by PI staining solution (Invitrogen) and analyzed by ow cytometry collecting 25,000 events per sample. The results were analyzed with FlowJo software
5
Cell cycle analysis via flow cytometry
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Cell cycle analysis was performed using propidium iodide (PI) staining as previously described [58 (link)]. Briefly, cells were incubated with five different concentrations of crizotinib (0.25 × IC50, 0.5 × IC50, 1 × IC50, 2 × IC50, or 4 × IC50) or media alone for 24, 48, and 72 h at 37 °C/5% CO2. Cells were then harvested and fixed in 80% ethanol at −20 °C. Cells were incubated in PI staining solution (Thermo Fisher Scientific, Dreieich, Germany) for 15 min at 4 °C and read on an Accouri C6 flow cytometer (Becton-Dickinson, Heidelberg, Germany). Total DNA content was measured on FL2-A [59 (link),60 (link)].
6
Cisplatin-Induced Cell Cycle Analysis
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Cells were transfected using shNUMB or control-shRNA for 72 h, then cells were plated in 60-mm dishes with or without cisplatin (10 µM) for 48 h. After trypsinized, cells were resuspended in PI staining solution (Thermo Fisher) according to manufacturer’s protocols. The percentage of cells in each phase of the cell cycle was determined by FACS flow cytometer (BD Biosciences). Analyses were performed separately, in triplicate.
7
Cell Cycle Analysis of AMO1 Cells
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The cell cycle perturbations of AMO1 cells were studied using Propidium iodide (PI) 24, 48, and 72 h post-treatment with different cynaropicrin concentrations (0.5, 0.9, 1.8, and 3.6 µM) or media alone. Cells were then harvested and fixed with 80% ethanol at −20 °C. After each specific incubation period, cells were incubated in PI staining solution (Thermo Fisher Scientific, Dreieich, Germany) at 4 °C. Fifteen minutes later, PI staining was measured using an Accuri C6 flow cytometer (Becton-Dickinson, Heidelberg, Germany). Total DNA content was detected on FL2-A [38 (link)].
8
Cell Cycle Analysis by Flow Cytometry
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JEG-3 or HTR-8/Svneo cells in logarithmic growth phase were planted in a 6-well plate at 1 × 106 cells/well. After transfection for 48 h, the cells were collected and fixed overnight at 4°C with pre-cooled 70% ethanol. On the next day, the fixed cells were collected and resuspended by adding 400 μL PBS including 100 μg/ml RNAase and 100 μg/ml PI staining solution (Thermo, USA). After staining for 30 min at the room temperature in the dark, the cells were assayed and analyzed using a BD FACSCalibur flow cytometer (BD Biosciences, USA).
9
Cell Cycle Analysis by Flow Cytometry
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Cell seeding in 6-well plates was performed at 2×105 cells per well. Following overnight culture, cells were irradiated, collected by trypsin treatment, and fixed with chilled 70% ethanol at −20 ℃ overnight. Next, the cells were resuspended in propidium iodide (PI) staining solution (eBioscience, San Diego, CA, USA) and submitted to >2 h incubation at 4 ℃ away from light. A total of 104 events were assessed per sample, and cell cycle distribution was evaluated on a BD FACSVerse with the ModFit LT software (Becton Dickinson and Co., Franklin Lakes, NJ, USA). Triplicate assays were carried out.
10
Cell cycle and apoptosis analysis
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For cell cycle analysis, cells were collected after transfection, then washed and fixed with cold 75% alcohol overnight. After wash with PBS, cells were labeled with propidium iodide (PI) (P4170-10, Sigma-Aldrich) and incubated at room temperature in the dark for 30 min. Cells were then filtered through a nylon mesh filter and subjected to flow cytometry (BD Biosciences). For cell apoptosis analysis, cells were harvested at 48 h after transfection, and stained using the FITC-Annexin V apoptosis detection kit and PI staining solution (88-8005-72, eBioscience, San Diego, CA) according to manufacturer's protocol. FACS (fluorescence activated cell sorter) analysis was performed within 4 h and the results were analyzed by FlowJo software (Version 7.6.1).
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