HeLa or HT1080 cells (200,000) were plated in 6-well plates in a volume of 2 mL and allowed to adhere 4-8 h. Prior to treatment, the media was removed and replaced and delmarine was added to the appropriate concentration (10 μM unless otherwise specified). The compounds were incubated for 18 h at which point the media was collected and the cells were trypsinized and harvested. The cells were centrifuged at 1000 × g for 5 min, washed with 3 mL of PBS, resuspended in 500 μL of PBS, and fixed by dropwise addition to 3 mL of 70% EtOH at −20°C. The fixed cells were centrifuged, washed once with 3 mL PBS, 500 μL of FxCycle PI/RNAse solution (ThermoFisher) was added, and the cells were incubated at room temperature for 30 min. The cells were analyzed by flow cytometry using a NovoCyte flow cytometer and 15,000 events were collected.
Fxcycle pi rnase solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FxCycle PI/RNase Solution is a ready-to-use cell cycle analysis reagent. It contains propidium iodide (PI) and RNase A, which are used to stain cellular DNA for flow cytometric analysis.
Automatically generated - may contain errors
Lab products found in correlation
Gallios cytometer, by Beckman Coulter (3 mentions)
Click it edu alexa fluor 488 flow cytometry assay kit, by Thermo Fisher Scientific (2 mentions)
Fcs express 6, by De Novo Software (1 mentions)
Novocyte facs, by Agilent Technologies (1 mentions)
Click it plus reaction cocktail, by Thermo Fisher Scientific (1 mentions)
13 protocols using fxcycle pi rnase solution
1
Cell Cycle Analysis of HeLa and HT1080 Cells
2
Cell Cycle Analysis by FACS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyze cell cycle changes by FACS, we harvested cells with trypsin and fixed with cold 70% ethanol. We stained fixed cells with propidium iodide using FxCycle PI/RNase solution (ThermoFisher Scientific), according to the manufacturer’s instructions. FACS was performed on three biological replicates with a FACS Accuri 6 machine and cell cycle was assessed on a minimum 15,000 gated cells, excluding cell debris and doublets, with FlowJo software (V10.1r5). See Supplementary Fig. 4D for example of gating strategy and Supplementary Data 1 for all counts and calculations.
3
OOS Effect on Cell Cycle and Division
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 266-6 cells were cultured in 6-well plates for 18 hours to study the effect of OOS in the cell cycle in vitro. Then, cells were treated with 1:100, 1:200, and 1:500 (V/Vf) dilutions of OOS in complete RPMI for 48 hours. The control cells were cultured with the control medium. Then, cells were trypsinized, washed once with phosphate-buffered saline (PBS), and fixed with 70% ethanol for 15 minutes at 4°C. Afterwards, cells were washed with PBS 3 times and incubated with propidium iodide (PI) containing FxCycle PI/RNase Solution (Thermo Fisher Scientific) following the manufacturer's indications. Finally, cell cycle changes were analyzed by flow cytometry using the Gallios cytometer (Beckman Coulter, Brea, Calif). For cell division analysis, 266-6 cells were fluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE) by incubating at 37°C for 30 minutes before seeding them into 6-well plates. Then, cells were treated as described above for cell cycle analysis. After 48 hours, cells were trypsinized, washed in PBS, and resuspended for flow cytometry studies by Gallios cytometer (Beckman Coulter).
4
Cell Cycle Analysis of PT2385 Treatment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cell proliferation assays, 1 × 106 cells from 10 μM PT2385-treated and sham-treated cells in normoxic and hypoxic conditions were fixed in 70% ethanol and washed with PBS. The cell pellet was resuspended in 0.5 mL of FxCycle PI/ RNAse solution (ThermoFisher, Carlsbad, CA) and incubated in the dark at room temperature for 30 min. Cells were analyzed for cell cycle fractions on a BD Accuri C6 Analyzer Flow Cytometer using 488-nm excitation and 585-nm collection filters. Data was analyzed using FCS express6 software from DeNovo (Glendale, CA).
5
Cell Cycle Analysis of URB447 Effects
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cell cycle analysis, 3 × 105 cancer cells were cultured in 6-well plates for 18 h in complete medium. The medium was changed and cells were treated with 10, 25 and 50 µM URB447 in fresh medium supplemented with 1% FBS for 24 h (0.1% DMSO was used as vehicle treatment for control cells). Attached cells were collected through trypsinization and subjected to phosphate-buffered saline (PBS) washes prior to fixation using 70% Ethanol for 30 min at 4 degrees. Ethanol was eliminated from cells through PBS washes prior to cell staining using propidium iodide (PI) containing FxCycle PI/RNase Solution (Thermo Fisher Scientific (Waltham, MA, USA)) following the manufacturer’s indications. Finally, the URB447 mediated perturbation of cell cycle was evaluated by flow cytometry using the Gallios cytometer (Beckman Coulter, Brea, CA, USA).
6
Cell Cycle Analysis of Melanoma Cells with Ocoxin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cell cycle analysis, 3 × 105 melanoma cells were cultured in 6-well plates for 18 h in complete medium. Cells were treated with 1:50 dilution of Ocoxin for 48 h in 1% FBS supplemented medium. Control cells were cultured with 1% FBS supplemented fresh medium without Ocoxin. Cells were then trypsinized, washed once with phosphate-buffered saline (PBS), and fixed with 70% ethanol for 30 min at 4 °C. Afterward, cells were washed with PBS twice and incubated with propidium iodide (PI) containing FxCycle PI/RNase Solution (Thermo Fisher Scientific, MA, USA) following the manufacturer’s indications. Finally, changes in the cell cycle were studied by flow cytometry using the Gallios cytometer (Beckman Coulter, Brea, CA, USA).
7
Cell Cycle Analysis of Neuroblastoma Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To follow the effects of the drugs on the cell cycle a FACS analysis was performed. All procedures were performed according to the protocol of the manufacturer (Invitrogen; Thermo Fisher Scientific, Inc.), SK-N-AS, SK-N-BE(2)-C, SK-N-DZ, SK-N-FI and SK-N-SH cells were collected 48 h after treatment with 0.5 and 1.0 MK-1775, 1.0 BMN673, 5.0 and 10.0 PD-0332991 and 5.0 and 10.0 µM BYL719, resuspended in cold PBS in 15-ml tubes and cold 70% ethanol was included dropwise while vortexing at a speed of 1,800 rpm for 10 sec at room temperature. Controls were made with PBS and DMSO at the tested concentrations. Subsequently, tubes were stored at 4°C until they were utilized. A total of 1×106 cells was analyzed per sample. Following centrifugation at 204 g for 10 min at 4°C, the pellet was dissolved in 0.5 ml FxCyclePI/RNAse solution according to the manufacturer's instructions (Invitrogen; Thermo Fisher Scientific, Inc. Stockholm, Sweden) at room temperature (without light) for 30 min before analysis using FACS Novocyte, Agilent and Flowjo_v10.8.1 software (Sapio Sciences, London, UK).
8
Cell Cycle Analysis of EWS-FLI1 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
U2OS cells were nucleofected with the dual promoter pCDH-EWS-FLI1-EF1a-mtagBFP (or empty vector) and analyzed at 48 hours. Cells were fixed using 4% formaldehyde for 15 minutes at room temperature. After washing with 1% BSA in PBS, cells were resuspended in 500μl of FxCycle™ PI/RNAse Solution (ThermoFisher Scientific) for 30 minutes at room temperature. Cell cycle profiles were also analyzed using Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Invitrogen). Cells were pulse labelled with 10μM EdU for 30 minutes and processed according to the manufacturer’s instructions. Flow cytometry was used to analyze the cell cycle profile for BFP positive, EWS-FLI1 (or empty vector) expressing cells.
9
Cell Cycle Analysis of EWS-FLI1 Expressing U2OS Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
U2OS cells were nucleofected with the dual promoter pCDH-EWS-FLI1EF1a-mtagBFP (or empty vector) and analyzed at 48 hours. Cells were fixed using 4% formaldehyde for 15 minutes at room temperature. After washing with 1% BSA in PBS, cells were resuspended in 500μl of FxCycle™ PI/RNAse Solution (ThermoFisher Scientific) for 30 minutes at room temperature. Cell cycle profiles were also analyzed using Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Invitrogen). Cells were pulse labelled with 10μM EdU for 30 minutes and processed according to the manufacturer’s instructions. Flow cytometry was used to analyze the cell cycle profile for BFP positive, EWS-FLI1 (or empty vector) expressing cells.
10
Cell Cycle Analysis by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells fixed in 70% ethanol were pelleted by centrifugation, washed with PBS and resuspended in 500 µL FxCycle PI/RNase solution (ThermoFisher Scientific, Altrincham, UK) according to the manufacturer’s protocol and analysed on FACS Symphony, gating for live cells (FSC-A vs. SSC-A), single cells (YG610/20-H vs. YG610/20-A) and DNA content (YG610/20-A vs. count). Data were processed to calculate percentage of cells in Sub-G1. G1/G0, S and G2/M using Floreada (https://floreada.io/ , accessed 3 April 2023).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!