The micronucleus assay was performed as described previously (Garaycoechea et al., 2018 (link)). Briefly, mice blood (8–12 weeks of age) was mixed with 100 μL PBS containing 1,000 U mL−1 of heparin (Calbiochem). Blood suspension was then added to 1 mL of methanol and stored at −80 °C overnight until further processing. 1 mL of fixed blood cells was washed with 6 mL of bicarbonate buffer (0.9% NaCl, 5.3 mM NaHCO3). Cells were suspended in 100 μL of bicarbonate buffer with 1 μL of FITC-conjugated CD71 antibody (Thermo Fisher) at 4 °C for 45 min. After centrifugation, pellets were washed with bicarbonate buffer and resuspended in 5 μg mL−1 PI/RNase Staining Solution (Thermo Fisher). Samples were analyzed immediately on an Nxt Attune FACS analyzer (Thermo Fisher) and data analyzed with FlowJo.
Pi rnase staining solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
PI/RNase Staining Solution is a fluorescent staining solution used for DNA content analysis in flow cytometry. It contains propidium iodide, a DNA-binding dye, and RNase to remove RNA interference. The solution allows for the measurement of cellular DNA content, which is useful for cell cycle analysis and DNA ploidy studies.
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Lab products found in correlation
Facscan flow cytometer, by BD (3 mentions)
Nxt attune facs analyzer, by Thermo Fisher Scientific (1 mentions)
Alamar blue dye, by Merck Group (1 mentions)
Annexin 5 dead cell apoptosis kit, by Thermo Fisher Scientific (1 mentions)
Aria 3 flow cytometer, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Guava easycyte ht, by Merck Group (1 mentions)
Click it edu alexa fluor 488 kit, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Cyan adp analyzer, by Beckman Coulter (1 mentions)
10 protocols using pi rnase staining solution
1
Micronucleus Assay for Mouse Blood
2
Evaluating Cell Viability and Apoptosis
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Cell viability was measured by cell count using trypan blue dye (Sigma, T8154) /hemocytometer or resazurin (Alamarblue dye, Sigma) with the Envision Fluorescent Reader (Perkin Elmer). Apoptosis was assessed using Annexin-V/Dead Cell Apoptosis Kit (V13241, ThermoFisher) and cell cycle analysis with PI/RNase Staining solution (F10797, ThermoFisher) as per manufactures’ instructions. Data were acquired using an LSRII flow cytometer and an Aria III flow cytometer (BD Biosciences, UK) and analyzed using FlowJo software (Tree Star Inc., USA).
3
Cell Cycle Analysis by Flow Cytometry
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Cells were cultured in 6-cm dishes and were harvested when grown to 80% confluence. Cells were washed with ice-cold D-Hanks buffer and fixed with 70% ethanol at 4 °C for 1 h. The cells were washed with ice-cold D-Hanks buffer one more time and stained with PI/RNase Staining Solution (Thermo Fisher Scientific). DNA data was analyzed by fluorescence-activated cell sorting (Guava easyCyte HT, Merck Millipore, Billerica, MA, USA).
4
Cell Cycle Progression Evaluation
5
Cell Cycle Analysis of CKD-602 Treatment
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The cells at a density of 2 × 105 cells/well in MEM/DMEM with 10% FBS were added to the wells of a 6-well plate and incubated overnight. Each cell was treated with 1/2 IC50 and IC50 CKD-602 for 48 h. Following treatment, cells were harvested by centrifugation at 1300 rpm for 3 min, washed twice in ice-cold PBS and fixed by incubating the cells overnight at − 20 °C with 70% ethanol. Cells were then washed with ice-cold PBS and resuspended in 500 μl PI/RNase staining solution (Invitrogen/ThermoFisher Scientific, Waltham, MA, USA). The cell cycle position was evaluated by FACS using an excitation laser set at 480 nm and a detection wavelength of 575 nm. A minimum of 10,000~30,000 events/sample was analyzed.
6
Cell Cycle Analysis of CCDC6, FBXO42 Knockouts
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Pure populations of cells transduced with an sgRNA targeting CCDC6, FBXO42, or ntc were grown in media treated with 1.2μM AZD6737 or an equivalent volume of DMSO for 24hr. Cells were washed and grown in fresh untreated media for another 24hr before harvesting for PI staining (total time post treatment = 48hr). To prepare for staining and flow cytometry, cells were washed in PBS (GIBCO) and cell pellets resuspended in ice-cold 70% EtOH while being vortexed. Fixation continued at −20°C for 1hr, then cells were washed in PBS and analyzed by flow cytometry to determine concentration. 5E5 fixed cells were spun down and resuspended in 500μL of PI/RNase staining solution (Invitrogen). Cells were stained for 30 minutes then analyzed by flow cytometry to determine DNA content distributions of each population.
7
Cell Cycle Analysis of T24 Cells
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After being treated with IC-2 (0–5 μM) and fixed with 10 ml cold ethanol overnight, T24 cells were washed in PBS and treated with 500 μl PI/RNase Staining Solution (Invitrogen, Carlsbad, CA, USA) containing DNase-free RNase and reagent for 30 min at 37°C. After incubation, the cells were washed and analyzed immediately by flow cytometry analysis using a Beckman-Coulter CyAN ADP Analyzer (Beckman, CA, USA). Data were analyzed with FlowJo Version 6.1 software (TreeStar, USA). Final data were calculated from at least 3 independent experiments.
8
Cell Cycle Analysis of WiDr Cells
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The WiDr cells (5 × 10 3 cells/well) were added to a 6-well plate which was incubated for 24 h for optimal growth. Subsequently, the cells were exposed to selected concentrations of PAL and PAA (1/5 IC 50 ) and incubated again 10 . Floating as well as attached cells were collected by adding 0.025% trypsin. The cells from each well were transferred to a separate eppendorf tube. 1 mL PBS was added and the PBS was removed with a micropipette and centrifuged at 2500 rpm for 5 min. The supernatant was removed and 1 μL RNase/PI staining solution (Thermo Fisher Scientific) was added and kept for 10 min a dark place (avoiding light) at 37°C. The cell cycle distribution was analysed using the FAC Scan Flow Cytometer (BD Biosciences) and the percentage of cells obtained in each cell cycle phase (G1-S and G2-M) was calculated using the software ModFit LT. 3.0 s for Windows (Verity Software House).
9
Cell Cycle Analysis of Anticancer Compounds
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The WiDr cells (5 × 10 3 cells/well) were added to a 6-well plate which was incubated for 24 h for optimal growth. Subsequently, the cells were exposed to selected concentrations of PAL and PAA (1/5 IC 50 ) and incubated again 10 (link) . Floating as well as attached cells were collected by adding 0.025% trypsin. The cells from each well were transferred to a separate eppendorf tube. 1 mL PBS was added and the PBS was removed with a micropipette and centrifuged at 2500 rpm for 5 min. The supernatant was removed and 1 µL RNase/PI staining solution (Thermo Fisher Scientific) was added and kept for 10 min a dark place (avoiding light) at 37°C. The cell cycle distribution was analysed using the FAC Scan Flow Cytometer (BD Biosciences) and the percentage of cells obtained in each cell cycle phase (G1/S and G2/M) was calculated using the software ModFit LT. 3.0 s for Windows (Verity Software House).
10
Cell Cycle Analysis of WiDr Cells
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The WiDr cells (5 × 10 3 cells/well) were added to a 6-well plate which was incubated for 24 h for optimal growth. Subsequently, the cells were exposed to selected concentrations of PAL and PAA (1/5 IC 50 ) and incubated again 10 (link) . Floating as well as attached cells were collected by adding 0.025% trypsin. The cells from each well were transferred to a separate eppendorf tube. 1 mL PBS was added and the PBS was removed with a micropipette and centrifuged at 2500 rpm for 5 min. The supernatant was removed and 1 μL RNase/PI staining solution (Thermo Fisher Scientific) was added and kept for 10 min a dark place (avoiding light) at 37°C. The cell cycle distribution was analysed using the FAC Scan Flow Cytometer (BD Biosciences) and the percentage of cells obtained in each cell cycle phase (G1-S and G2-M) was calculated using the software ModFit LT. 3.0 s for Windows (Verity Software House).
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