To perform cell cycle analysis, cells were fixed with 70% ethanol at −20°C and stained with PI solution (Life Technologies) in the presence of 62 μg/ml RNase A, followed by flow cytometric analysis using BD Accuri flow cytometer (BD biosciences, San Jose, CA).
Pi solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
PI solution is a reagent used in flow cytometry and other cell analysis applications. It is a fluorescent dye that binds to DNA, allowing for the detection and quantification of cellular DNA content. The solution is commonly used to stain cells for analysis of cell cycle, cell proliferation, and apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Accuri flow cytometer, by BD (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Rnase a, by Thermo Fisher Scientific (2 mentions)
Lsm 7 duo, by Zeiss (1 mentions)
Zen 2012, by Zeiss (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Flow cytometry, by BD (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Annexin 5 fitc solution, by Thermo Fisher Scientific (1 mentions)
Facscan, by BD (1 mentions)
Annexin 5, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Axiovert 25, by Zeiss (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Accur c6 flow cytometer, by BD (1 mentions)
Rnase a, by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
Pi solution, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
27 protocols using pi solution
1
Cell Cycle Analysis by Flow Cytometry
2
Quantifying Root Quiescent Center Cells
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Confocal images of root z-stacks (2 μm sections) were obtained using a Zeiss LSM 7 DUO confocal laser scanning microscope (Carl Zeiss, Germany), with a 40× oil immersion objective. At least 5 seedlings from three independent experiments (total of 15 seedlings) were analyzed. Roots were stained with 10 μg/ml PI solution (Life Technologies, USA) for 1 to 10 min. Fluorescence intensity was acquired at 488/505–530 nm excitation and emission for GFP, 543/560 nm for PI, and 543/560–615 nm for Alexa Fluor 555. ZEN2012 software (Carl Zeiss, Germany), Image J (http://rsbweb.nih.gov/ij ), and Photoshop were used to analyze the green fluorescence intensity. QC cell dimensions and area were measured using Image J. For measurement of QC cell dimensions, the longest sideward and upward distances between the plasma membrane were chosen as length and height, respectively. QC cell area was measured as area enclosed by free hand line drawn over the PI-stained plasma membrane of QC cells.
3
Cell Cycle Profiling of OS Cells
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In order to examine the effects of auranofin and its combination with vorinostat and rapamycin on cell cycle profiles of OS cells, we performed PI-staining and flow cytometry analysis. Cells were fixed overnight with 70% ethanol at -20°C and stained with PI solution (Life Technologies) in the presence of 62 μg/ml of RNase A, followed by flow cytometric analysis using BD Accuri flow cytometer (BD biosciences, San Jose, CA). Data was analyzed using the FlowJo V10 software (FlowJo LLC, Ashland, Oregon).
4
Oxidative Stress Measurement in MSC80 Cells
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Flow cytometry experiments were performed on a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA). Cellular oxidative stress was measured by flow cytometry using dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay (Molecular Probes by Life technologies, D-399). MSC80 cells (250000 cells/well in 6-well plates) were pretreated with T0 for 72 h followed by 2 h of tert-butyl hydroperoxide (tBHP 20 µM) treatment to induce ROS production. After tBHP treatment, the cells were washed, collected and incubated with 0.5 µM DCFH-DA for 30 min at 37 °C. After DCFH-DA incubation the cells were centrifuged and resuspended again in 1 ml of PBS with 1 µl of PI solution (50 µg/ml, Life technologies) followed by flow cytometry. A total of 10.000 events were recorded with a flow rate of less than 200 cells/second for each assay. Data analysis was performed using BD Cell Quest Pro Software.
5
Quantifying Cell Death Using Hoechst and PI
6
Cell Cycle Analysis of A2780/PTX Cells
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A2780/PTX cells (5.0 × 104 cells/well) were seeded in 6-well plates and treated with blank LPN (250 μg/mL), free PTX+TET, P/LPN+T, P/iRGD LPN + T, P+T/LPN, or P+T/iRGD LPN at equivalent concentrations of both PTX and TET at 10 μM for 48 h. The cells were harvested and washed with PBS and were then fixed in cold 70% ethanol and stored at −20 °C overnight. The cells were collected by centrifugation and stained with 5 μL of PI solution (Life Technologies, USA) for 10 min. The cell cycle results were obtained using flow cytometry (BD Biosciences, California, USA), and the cell distributions were analyzed using ModFit LT software (version 3.0, USA).
7
Live-Dead Cell Staining Protocol
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The live/dead staining was performed 48 hours after cell seeding as was described previously61 (link). Hoechst 33258 (Sigma) dissolved in PBS and propidium iodide (PI) solution (Life Technologies) were added directly to cell culture medium at final concentrations of 2 μg/ml and 5 μg/ml respectively. Cells were incubated for 2 hours and then immediately examined with an inverted fluorescence microscope.
8
Quantifying Cell Death in Cultured Cells
9
Quantifying Apoptosis in KGN Cells
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Flow cytometry analysis and Annexin V-FITC Apoptosis Detection Kit were performed to detect the cell apoptosis. After transfected for 48 h, KGN cells were harvested, were washed and resuspended. Annexin V-FITC solution (Invitrogen, USA) with a concentration of 0.25 μg/mL and PI solution (Invitrogen, USA) with a concentration of 1 μg/mL were added into cell suspension and incubate for 15 min in the dark. Finally, cell apoptosis was analyzed by a FACScan instrument (Becton Dickinson, Franklin Lakes, NJ, USA).
10
Apoptosis Assay Using miR-375-3p
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AMC-HN-8 and Tu-212 cells were seeded into 6-well plates (1×105 cells/well) and transfected with miR-375-3p mimics or inhibitor. After 48 h, cells were collected, washed twice with PBS and stained with Annexin V (Invitrogen; Thermo Fisher Scientific, Inc.) for 15 min in the dark at room temperature. Cells were subsequently stained with propidium iodide (PI) solution (Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 1 min. The apoptotic cells were analyzed using a flow cytometer (BD FACSCalibur; BD Biosciences) and FlowJo software version 10.6 (FlowJo LLC).
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