HeLa cells were seeded in 6‐well plates at a density of 4 × 105 cells per well for 24 h. Following the attachment, the cells were treated by free ARV‐771 or ARV@PDSA at an ARV‐771 concentration of 1000 nm for another 24 h. Then, the cells were harvested with 0.25% trypsin, washed once with cold PBS, and placed in precooled 70% ethanol at −20 °C for 24 h for fixation. Then, the cells were washed with PBS once and incubated with propidium iodide (50 µg mL−1, Thermo Fisher Scientific, F10797) in a dark environment at room temperature for 30 min. Subsequently, the cells were analyzed using a flow cytometer (BD, LSRFortessa) and the data were analyzed using FlowJo software, version 8.8.4 (Tree star, Inc., Ashland, Or, USA).
F10797
Manufactured by Thermo Fisher Scientific
The F10797 is a laboratory instrument manufactured by Thermo Fisher Scientific. It is designed for the analysis and processing of samples in a research or industrial setting. The core function of this product is to perform specific analytical tasks, although the exact details of its intended use are not provided.
Automatically generated - may contain errors
3 protocols using f10797
1
Cell Cycle Analysis of ARV-771 Treatment
2
Cell Cycle Progression Analysis
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For cell-cycle analysis, cells were plated in 10-cm cell culture dishes at a density of 2 mol/L cells per plate in biological triplicate (approximately 70% confluency) and treated the next day. Cells were then treated with 2 mmol/L of Thymidine (Sigma-Aldrich T1895) and incubated for 24 hours to allow block of the cells in the S phase of the cell cycle. Cells were then released from the Thymidine block by replenishing with regular media for 6.5 hours to allow cells to reengage in cell-cycle progression. Following the 6.5 hours release, cells were then treated with either DMSO or 20 μmol/L SMAP and incubated for 6, 10, and 16 hours and subsequently harvested and fixed using 70% cold ethanol. PI (Thermo Fisher Scientific F10797) staining was performed according to FxCycle PI/RNAse Staining Solution protocol available online (www.thermofisher.com ) and cell-cycle profiles were registered using the Bio-Rad Zed flow machine. Cell-cycle graphs were finalized using FlowJo 8.
3
TNF-α and DKK1 Impact on Apoptosis and Cell Cycle
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NP cells were stimulated with TNF‐α and DKK1, either alone or in combination with the transfection of miR‐640 mimics or a miR‐640 inhibitor, and then cultured at 37°C with 5% CO2 in humid air. The cells were washed twice with PBS and centrifuged. For apoptosis detection, discarded the supernatants and the cells were resuspended in 1 × annexin‐binding buffer. The apoptosis rate was detected by staining with Annexin V‐FITC and propidium iodide (Millipore, APOAF‐50TST). For cell cycle analysis, the cells were fixed in 70% ethanol overnight and underwent propidium iodide staining (Thermo, F10797). Finally, all samples were detected on FC500 flow cytometry (Beckman).
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