HS01 cells were seeded at 250,000 cells/well in 6-well CellBIND (Corning) plates, grown to ~80% confluency, and treated with CUDC907 for 24 h. EdU (10 μM; Click-iT EdU kit; Molecular Probes, ThermoFisher) was added during the last 3 h. Cells were harvested with 0.05% trypsin, stained with violet live/dead stain (ThermoFisher), and permeabilized. DNA labeling with FxCycle stain (ThermoFisher) was conducted according to manufacturer’s protocol. Cell populations were identified by flow cytometry on CytoFlex (Beckman Coulter) and analyzed by CytExpert software (Beckman Coulter).
Fxcycle stain
Manufactured by Thermo Fisher Scientific
FxCycle stain is a fluorescent DNA stain used in flow cytometry applications to detect and quantify cellular DNA content. It binds stoichiometrically to DNA, allowing for the determination of cell cycle and apoptosis status.
Automatically generated - may contain errors
Lab products found in correlation
Click it edu kit, by Thermo Fisher Scientific (5 mentions)
Live dead violet stain, by Thermo Fisher Scientific (3 mentions)
Cytoflex, by Beckman Coulter (3 mentions)
Cytexpert software, by Beckman Coulter (3 mentions)
Phospho rb ser807 811, by Cell Signaling Technology (2 mentions)
Cellbind, by Corning (1 mentions)
Lsrii flow cytometer, by Tree Star (1 mentions)
6 protocols using fxcycle stain
1
Cell Proliferation Assay with EdU
2
Quantify Cell Cycle Dynamics in Mouse Tumors
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Post-natal P10 G-Smo mice were injected IP with CT-179 (80 mg/kg), or POx-Palbo (25 mg/kg, every day from P10 to P13), or combination (CT-179 + POx-Palbo, EOD from P14) or saline control solution and tumors were harvested after 6 or 24 hours. EdU (40 mg/kg) was administered IP 30 minutes before harvest. Tumors were dissociated using the Worthington Papain Dissociation System Kit, then fixed for 15 minutes on ice and washed with FACS Wash buffer (2% FBS in PBS). Fixed cells were stained with fluorophore markers for DNA (FX Cycle stain, Thermo Fisher Scientific), EdU (Click-it Edu Kit, Thermo Fisher Scientific), and phospho-RB content (phospho-RBSer807/811, Cell Signalling Technology) as previously described [59 (link)]. Stained cells were resuspended in sheath fluid and ran on LSR II flow cytometer for 50000 events at the UNC Flow Cytometry core, using appropriate compensation controls and analyzed using FlowJo® software (Tree Star).
3
Tumor Cell Proliferation Profiling
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P10 to P12 tumor mice were administered drugs [POx-Palbo, Palbo-HCl, POx-Sapanisertib, and POx-(Palbo+Sapanisertib)] via intraperitoneal injections. EdU (40 mg/kg) was administered to mice 30 min before harvest. Tumor tissue was harvested and subjected to cellular dissociation via Worthington Papain Dissociation System kit. Dissociated tumor cells were fixed for 15 min on ice and washed with fluorescence-activated cell sorting wash buffer. Fixed cells were stained with fluorophore markers for DNA (FX Cycle stain; catalog no. F10347, Thermo Fisher Scientific), EdU (Click-it Edu Kit; catalog no. C10337, Thermo Fisher Scientific), and pRB content [phospho-RB (Ser807/811); catalog no. 8974S, Cell Signaling Technology]. Stained cells were prepared in appropriate strained for flow analysis.
4
Fibroblast Synchronization for Cell Cycle Analysis
5
Cell Cycle Analysis of Drug Treatments
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HS01 cells were seeded at 125,000 cells/well in a 6-well CellBIND Corning plate and al-lowed to attach overnight. Cells were then treated with 0.001% DMSO, 2.5 µM pictilisib, 150 nM PF-3748309, or a combination of the two drugs for 24 h. EdU (10 μM) (Click-iT EdU kit; Molecular Probes, ThermoFisher) was added during the last 3 h. Cells were collected with 0.05% trypsin, stained with violet live/dead stain (ThermoFisher Scientific), and permeabilized. DNA labeling with FxCycle stain (ThermoFisher Scientific) was performed according to manufacturer’s instructions. Cell populations were identified by flow cytometry on CytoFlex (Beckman Coulter, Brea, CA, USA) and analyzed by CytExpert software (Beckman Coulter). Experiment was performed in three replicates.
6
Cell Proliferation Assay with Pictilisib and PF-3748309
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HS01 cells were seeded at 125,000 cells/well in a 6-well CellBIND Corning plate and al-lowed to attach overnight. Cells were then treated with 0.001% DMSO, 2.5 μM pictilisib, 150 nM PF-3748309, or a combination of the two drugs for 24h. EdU (10 μM) (Click-iT EdU kit; Molecular Probes, ThermoFisher) was added during the last 3h. Cells were collected with 0.05% trypsin, stained with violet live/dead stain (ThermoFisher Scientific), and permeabilized. DNA labeling with FxCycle stain (ThermoFisher Scientific) was performed according to manufacturer’s instructions. Cell populations were identified by flow cytometry on CytoFlex (Beckman Coulter, Brea, CA, USA) and analyzed by CytExpert software (Beckman Coulter).
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