The cells were collected after 72 h of treatment, washed twice with phosphate‐buffered saline (PBS) solution, and suspended in 500 μl of binding buffer solution. Propidium iodide (PI) and annexin V‐FITC (BD Biosciences) were added, and the mixture was incubated for 15 min at room temperature. Apoptotic changes were detected using a flow cytometer (BD Biosciences). Cells were fixed by adding 70% anhydrous ethanol solution after discarding the supernatant and stored at −20°C overnight to prepare for cell cycle analysis. Before detection, the cells were stained with PI and RNase A (Elabscience) for 30 min at room temperature.
Rnase a
Manufactured by Elabscience
Sourced in China
RNase A is a ribonuclease enzyme that catalyzes the degradation of single-stranded RNA. It is commonly used in molecular biology applications to remove unwanted RNA from DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 fitc, by BD (1 mentions)
Propidium iodide, by BD (1 mentions)
Accuri c6 plus software, by BD (1 mentions)
Facscan flow cytometry system, by BD (1 mentions)
Anti cd29 fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd44 apc, by Novus Biologicals (1 mentions)
Anti cd45 apc, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Elabscience (1 mentions)
4 protocols using rnase a
1
Apoptosis and Cell Cycle Analysis
2
Cell Cycle Analysis of Cancer Cells
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The exponentially growing SCC-15 and Cal-27 cells were incubated in 6-well plates with a concentration of 3 × 105/ well and allowed to adhere overnight. After incubation with different doses (0, 10, 15, 20 nM) of ID09 or paclitaxel (20 nM for positive control) for 24 hours, cells were harvested and fixed in ice-cold 70% ethanol overnight at 4°C, which was followed by centrifugation and rinse with ice-cold PBS. Subsequently, the cells were reacted with PI and RNase A (Elabscience, China) in dark at room temperature for 30 minutes. Finally, the cell cycle was acquired using a flow cytometer, and the data were analyzed with BD Accuri C6 plus software (Becton Dickinson, Franklin Lakes, NJ, USA).
3
Cell Cycle and Apoptosis Analysis
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Flow cytometry was performed to detect cell cycle and cell apoptosis. For cell cycle, cells were harvest and stained with propidium iodide (PI) and RNase A (Elabscience, Wuhan, China) for 1 h before being fixed with 70% cold ethanol for 2 h. For cell apoptosis, cells were stained with PI and Annexin V-FITC/APC (Elabscience, Wuhan, China). The stained cells were detected using a flow cytometer (Beckman, CA, USA).
4
Characterization of Mesenchymal Stem Cells
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To confirm surface marker of MSCs, flow cytometry analysis was applied according to the manufacturer’s instructions. 1 × 106 cells at P3 in the logarithm growth period were collected. After washing with 1% pre-cooled FBS/PBS and centrifuging at 350×g for 5 min, these cells were incubated with anti-CD45-APC (Invitrogen, USA), anti-CD29-FITC (Invitrogen, USA), and anti-CD44-APC (Novus Biologicals, USA) in the dark at 4 °C for 30 min, respectively. Labeled cells were washed twice and examined using the FACScan flow cytometry system (BD, Franklin Lakes, USA). FlowJo software (TreeStar, Ashland, OR, USA) was used to analyze the data. PBS solution was used as negative control. For cell cycle analysis of DNA content, the cells were cultured for 48 h under normoxia or hypoxia condition before they were collected, washed with PBS, and resuspended with 0.3 ml PBS and 1.2 ml pre-cooled 100 % ethanol for 1 h at − 20 °C. The cells were then centrifuged (300×g, 5 min) and resuspended with 1 ml PBS for 15 min at room temperature. The cells were then centrifuged (300×g, 5 min) again. One hundred microliters RNase A (Elabscience Biotechnology Co., Ltd., China) was added to each sample which was incubated at 37 °C for 30 min. Before test, 400 μl propidium iodide (Elabscience Biotechnology Co., Ltd., China) was added to each tube at 4 °C for 30 min.
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