Three CRC cell lines were all plated and cultured on 6-well plates for 24 h and treated with the low (IC25), medium (IC50), and high concentrations (IC75) of ECD for 48 h. For the cell cycle assay, the cells (at least 1 × 106) were fixed by 75% ethanol and stored at 4°C overnight. After washing twice with cold PBS, 300 μl PI/RNase staining (#550825, BD Biosciences, United States) was added to each sample and incubated for 30 min at 37°C under protection from light. The cell cycle was then analyzed by flow cytometry (FACSCalibur; Becton Dickinson, United States). Similarly, for the cell apoptosis assay, both floating and attached cells in each well were harvested and washed twice with cold PBS. Then, cells were incubated with 3 μl FITC-Annexin V (#556419, BD Biosciences, United States) and 5 μl PI (#556463, BD Biosciences, United States) in 100 μl binding buffer at 37°C under protection from light. Next, 200 μl binding buffer was added to each sample, and cell apoptosis was also measured by flow cytometry.
Pi rnase staining
Manufactured by BD
Sourced in United States
The PI/RNase staining is a laboratory technique used to measure DNA content in cells. It utilizes propidium iodide (PI) and RNase to selectively stain and quantify the DNA present in a sample.
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6 protocols using pi rnase staining
1
Cell Cycle and Apoptosis Analysis
2
Cell Cycle Analysis of HSFs
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HSFs stimulated with ADSC-Exo or PBS in six-well cell culture plates were digested with 0.25% trypsin and subjected to centrifuge at 1000 rpm for 5 min. Then, the pellets were washed with PBS, cautiously added, dropwise with precooled 75% ethanol to make HSFs be fixed uniformly. HSFs were cryopreserved at − 20 °C at least for 2 h. Thereafter, HSFs were washed with PBS twice at 1500 rpm for 10 min, resuspended with 200 μl of PI/Rnase staining (BD, Biosciences), and incubated in the dark places for 15 min at room temperature. The percentage of cell cycle in each phase was detected by using BD Accuri™ C6 flow cytometer.
3
Cell Cycle Analysis of Drug Treatments
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Cells were seeded in a 12-well plate and treated with indicated drugs (DMSO, 0.1 μM, 0.5 μM, 1.0 μM, 2.0 μM BI 2536) for three days. Then cells were washed with PBS, harvested, and fixed with anhydrous ethanol overnight. After washed with PBS, PI/RNase staining (550825, BD Biosciences) was added to suspend cells and incubate for 30 minutes in dark. The cell cycle distribution was determined by flow cytometry (BD Biosciences).
4
Cell Proliferation and Cell Cycle Analysis
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After transient transfection for at least 48 h, 1 × 103 cells were cultured in 96-well plate with 200 µl of medium. The cell proliferation assays were measured by CCK-8 (MedChemExpress, USA) at a 1:10 dilution with serum-free medium every 24 h for four days directed by the manufacturer’s protocols. Finally, OD450 of cells over four days was shown by GraphPad Prism to reflect the ability of cell proliferation. As for cell cycle analysis, Caki-1 cells were labeled with PI/RNase Staining (BD Bioscience) Buffer after transfected with Si-CENPA for 48 h. The DNA content was measured using flow (Beckman FC500, USA) cytometry and displayed by Modfit software.
5
Cytotoxicity and Cell Cycle Effects of KR-12-a5 on HBMSCs
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The cytotoxicity of KR-12-a5 (GL Biochemistry, Shanghai, China) was assessed by a CCK-8 test (Donjindo, Japan). Briefly, various concentrations of KR-12-a5 (0, 1, 2, 4, 8, 16, 32, or 64 μg/ml) were added into 96-well plates plated with HBMSCs (5.0 × 103 cells/well) for 1, 3, or 5 days. At each time point, the cell culture medium was removed and 100 µl of 10% CCK-8 solution was added to each well. The absorbance of the solution was tested at 450 nm after 2 h.
PI/RNase staining (BD Biosciences, San Diego, CA, USA) was used to test the cell cycle of HBMSCs after KR-12-a5 incubation. Briefly, after 48 h of KR-12-a5 treatment, HBMSCs were fixed with 75% ethanol at −20°C for 2 h. After fixation, the cells were resuspended in 0.5 ml of PI/RNase staining buffer and incubated for 15 min. The cell cycle proportions (G0, G1, S, and G2-M phases) were tested by a flow cytometer.
Apoptosis was assessed using flow cytometry while double-staining for Annexin V-FITC and PI (BD Biosciences, San Diego, CA, USA). HBMSCs were collected after 48 h of KR-12-a5 treatment and stained with 200 μl of 1× binding buffer containing 5 μl of Annexin V-FITC and 10 μl of PI for 15 min.
PI/RNase staining (BD Biosciences, San Diego, CA, USA) was used to test the cell cycle of HBMSCs after KR-12-a5 incubation. Briefly, after 48 h of KR-12-a5 treatment, HBMSCs were fixed with 75% ethanol at −20°C for 2 h. After fixation, the cells were resuspended in 0.5 ml of PI/RNase staining buffer and incubated for 15 min. The cell cycle proportions (G0, G1, S, and G2-M phases) were tested by a flow cytometer.
Apoptosis was assessed using flow cytometry while double-staining for Annexin V-FITC and PI (BD Biosciences, San Diego, CA, USA). HBMSCs were collected after 48 h of KR-12-a5 treatment and stained with 200 μl of 1× binding buffer containing 5 μl of Annexin V-FITC and 10 μl of PI for 15 min.
6
Cell Cycle Analysis via PI/RNase Flow Cytometry
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Flow cytometry with PI/RNase staining (BD Biosciences, San Jose, CA, USA) was used to detect the effects of PTE in cell cycle distribution. Briefly, T98G, LN18, LN229, U87, and C6 glioma cells were seeded in 6-well plates and cultured 8 h. Then, the cells were treated with PTE (20, 40, and 80 µM) and harvested for 48 h. Cells were fixed with 70% ethanol overnight at − 20 °C and incubated with PI/RNase in the dark for 15 min. Stained cells were subsequently analyzed with a flow cytometer (Becton Dickinson, San Diego, CA, USA).
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