The analysis of cell cycle progression affected by As2O3 exposure was done in the case of both long-term as well as short-term exposure. The MCF-10A_Ctr and MCF-7_Ctr were used as controls for calibration. For the long-term evaluation the MCF-10A_As2O3_3w, MCF-10A_As2O3_6w, MCF-7_As2O3_3w and MCF-7_As2O3_6w were tested. For short-term exposure evaluation, MCF-10A cells (both controls and long-term intoxicated) were exposed to 2.5 μM of As2O3 for 24h, while the MCF-7 cell (all experimental settings) were exposed to 5 μM of As2O3. The difference in tested concentrations was because the MCF-10A cell lost viability when exposed to 5 μM of As2O3. The cells were recovered from culture and fixed with 1 mL of 70% ethanol solution. After 45 minutes of incubation at 4 °C, the cells were washed with PBS. The samples were then incubated at RT for additional 15 minutes with 500 μL of RNase buffer from Thermo Fisher Scientific (Waltham, MS, USA). For DNA staining, a propidium iodine (PI) solution of 2 mg/mL was added. After 30 minutes, the samples were pelleted and resuspended in 500 μL flow cytometry wash solution. Reading was done on the BD FACS Canto II Flow cytometer (San Jose, CA, USA).
Rnase buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RNase buffer is a solution designed for the inactivation and removal of RNase enzymes. It provides a simple and effective way to protect RNA samples from RNase-mediated degradation during various molecular biology workflows.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2 flow cytometer, by BD (1 mentions)
Annexin 5, by Thermo Fisher Scientific (1 mentions)
Facsdiva version 6, by BD (1 mentions)
Propidium iodine, by Thermo Fisher Scientific (1 mentions)
Pbs 1x, by Merck Group (1 mentions)
Pbs 1x, by Thermo Fisher Scientific (1 mentions)
Annexin 5 binding buffer, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
2 protocols using rnase buffer
1
Cell Cycle Progression Analysis of As2O3 Exposure
2
Cell Cycle and Apoptosis Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The miRNA transfected and the control cell suspension was washed three times with PBS 1X (Merck KGaA, Darmstadt, Germany) and fixed with pre-chilled 70% ethanol for 50 min for the analysis of cell cycle progression. Afterward, the cells were rewashed with PBS 1X and treated with RNase buffer (40 µg/mL RNase and 0.2% BSA) (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min, at RT. At the end of this time, the propidium iodine (Thermo Fisher Scientific, Waltham, MA, USA) was added in dark conditions at a final concentration of 10 µg/mL in 500μL buffer and incubated at RT for another 30 min.
For apoptosis/necrosis ratio analysis at flow cytometry, the miRNA transfected, and control cells were trypsinized, resuspended in complete media and washed 3 times with PBS 1X. After the washing step the cells were centrifuged at 1200 rpm for 5 min and mixed with Annexin V Binding buffer (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA). The cells were then stained with 5 μL Annexin V (Thermo Fisher Scientific, Waltham, MA, USA) and 5 μL propidium iodide (Merck KGaA, Darmstadt, Germany) for 15 min.
The samples were read at the flow cytometer BD FACS Canto II instrument (BD, San Jose, CA, USA). The initial gaiting was fixed based on the control for each cell line. The data was analyzed with FACS Diva version 6.0 software (BD, San Jose, CA, USA).
For apoptosis/necrosis ratio analysis at flow cytometry, the miRNA transfected, and control cells were trypsinized, resuspended in complete media and washed 3 times with PBS 1X. After the washing step the cells were centrifuged at 1200 rpm for 5 min and mixed with Annexin V Binding buffer (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA). The cells were then stained with 5 μL Annexin V (Thermo Fisher Scientific, Waltham, MA, USA) and 5 μL propidium iodide (Merck KGaA, Darmstadt, Germany) for 15 min.
The samples were read at the flow cytometer BD FACS Canto II instrument (BD, San Jose, CA, USA). The initial gaiting was fixed based on the control for each cell line. The data was analyzed with FACS Diva version 6.0 software (BD, San Jose, CA, USA).
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!