Cells were harvested by 0.25% trypsin without EDTA (Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA) 48 h after transfection. For the apoptosis assay, 1×106 transfected cells were resuspended in 100 μL of binding buffer and labeled with 5 μL of Annexin V and 5 μL of 7-AAD according to the protocol using the PE Annexin V Apoptosis Detection Kit I (BD biosciences, CA, USA). Flow cytometry assays were performed to detect the cell apoptosis ratio by FACStation (FV500, Beckman Coulter, Brea, CA, USA).
Facstation
Manufactured by Beckman Coulter
Sourced in United States
The FACStation is a computer workstation designed for use with Beckman Coulter flow cytometry systems. It provides a platform for data acquisition, analysis, and management of flow cytometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Pe annexin 5 apoptosis detection kit 1, by BD (3 mentions)
Pi rnase staining buffer, by BD (3 mentions)
Propidium iodide, by Merck Group (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Kaluza flow analysis software, by Beckman Coulter (1 mentions)
Dna staining solution, by MultiSciences Biotech (1 mentions)
Facstation, by BD (1 mentions)
Apc annexin 5 kit, by BD (1 mentions)
Propidium iodide, by BD (1 mentions)
Dead cell apoptosis kit, by Thermo Fisher Scientific (1 mentions)
Annexin 5 alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
7 protocols using facstation
1
Measuring Apoptosis in Transfected Cells
2
Cell Cycle Analysis by Flow Cytometry
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For cell cycle analysis, 2×106 cells were harvested, fixed with 3 ml of cold 75% ethanol at −20°C overnight, and washed twice with PBS. The cells were then resuspended in 500 µl of PBS and simultaneously stained with 200 µl of DNA staining solution (MultiSciences, Hangzhou, China) at 25°C for 30 min. The percentage of cells in each cell cycle phase was determined using a FACStation (FV500; Beckman Coulter, La Brea, CA, USA) and analyzed using Kaluza Flow Analysis software (Beckman Coulter, Inc.).
3
Cell Cycle and Apoptosis Analysis
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For cell cycle analysis, 2 × 106 cells were harvested, fixed with 4 mL of cold 75% ethanol at −20°C overnight, and washed twice with PBS. The cells were then resuspended in 500 μL of PBS and simultaneously stained with 200 μL of propidium iodide (50 μL/mL; Sigma-Aldrich) and incubated with 20 μL of RNase (1 mg/mL; Sigma-Aldrich) in a 37°C water bath for 15–20 min. The percentage of cells in each phase of the cell cycle was determined using a FACStation (FV500, Beckman Coulter, Brea, CA, USA) and analyzed using the Kaluza® Flow Analysis Software. Each experiment was repeated in triplicate.
For apoptosis analyses, 1 × 105 cells were stained with Annexin V and propidium iodide using an Annexin V-APC kit (BD Pharmingen™ catalog number 550474, BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with a FACStation equipped with CellQuest software. Each experiment was repeated in triplicate.
For apoptosis analyses, 1 × 105 cells were stained with Annexin V and propidium iodide using an Annexin V-APC kit (BD Pharmingen™ catalog number 550474, BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with a FACStation equipped with CellQuest software. Each experiment was repeated in triplicate.
4
Evaluating Apoptosis and Cell Cycle
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CCs were plated in 24-well plates at 2 × 105 cells per well for 24 h and were incubated in culture medium with 10 nM RvE1 for an additional 8 h. Washed with PBS, cells were prepared for apoptosis with the PE Annexin V Apoptosis Detection Kit I (BD Biosciences, New Jersey, USA). As for cell cycle assays, we fixed cells with 70% ethanol and stained them with PI/RNase Staining Buffer (BD Biosciences, New Jersey, USA) after incubation at 4°C overnight. FAC Station (FV500, Beckman Coulter, Brea, USA) was utilized to detect cell apoptosis ratio and cell cycle profile, and Flow Jo software (version 10.4, Flow Jo, Oregon, USA) was used to analyze data.
5
Cell Apoptosis and Cycle Analysis
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PHGDH siRNA and a negative control siRNA were transfected as mentioned above.For cell apoptosis analysis, cells were prepared with the PE Annexin V Apoptosis Detection Kit I (BD Biosciences, New Jersey, USA) according to the manufacturer’s recommendations. For cell cycle analysis, cells were fixed and permeabilized by 75% ethanol, and were stained by PI/RNase Staining Buffer (BD Biosciences, New Jersey, USA) after incubation at − 20 °C overnight. The cell apoptosis ratio and cell cycle profile were detected by FAC Station (FV500,Beckman Coulter, Brea, USA), and the raw data were analyzed using FlowJo 10.0.7 software (FlowJo, Oregon, USA).
6
Apoptosis and Cell Cycle Analysis
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Cells were harvested 48 hours after transfection. For apoptosis analysis, the transfected cells were stained using the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 and propidium iodide (PI) (Invitrogen, catalog number V13241) according to the manufacturer’s recommendations. For cell cycle analysis, after fixing and permeabilizing the transfected cells, 0.5 ml of PI/RNase Staining Buffer (BD Biosciences, USA, catalog number 550825) was used per sample (1 × 106 cells) and incubated for 30 minutes at room temperature. Thereafter, the cell apoptosis ratio and the percentage of cells in each phase of cell cycle were measured using a FACStation (FV500, Beckman Coulter, Brea, CA, USA). The results were analyzed using FlowJo 7.6.2 software.
7
Cell Cycle Analysis by Flow Cytometry
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Cells (1–2 × 106) were harvested, washed twice with 1 × PBS. The cells were fixed with 4 ml of cold 75% ethanol at −20°C overnight and then washed twice with 1 × PBS. The cells were then resuspended in 500 μl of 1× PBS and stained with 200 μl of propidium iodide (50 μl/ml; Sigma-Aldrich, Missouri, US) and 20 μl of RNase (1 mg/ml; Sigma-Aldrich, Missouri, US) in a 37°C water bath for 15 to 20 minutes. Cell cycles were determined by FAC Station (Beckman Coulter, FV500) and analyzed by using Kaluza® Flow Analysis Software and a published method. The assays were repeated three times.
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