When transfected SKOV3 and OVCAR3 cells grew to 80% confluence, the cells in suspension were collected, and the cells on the dish were digested into single-cell suspension by trypsin (Hyclon, Logan, Utah, USA). All cells were collected and mixed with 5 μL Annexin V-FITC and 5 μL propidium iodide (PI) solution (KeyGEN, Nanjing, China) for 15 min at room temperature in the dark. Apoptosis rates were determined by a BD flow cytometer (Franklin Lakes, NJ, USA). B3 quadrant represented viable cells and B2 and B4 quadrants represented apoptotic cells. For detection of cell cycle distribution, the transfected cells were digested, washed, and incubated with 1 μl RedNucleus I (Sysmex Corporation, Kobe, Japan) at 37 °C for 10 min and then measured the cell cycle distribution with a BD flow cytometer (Franklin Lakes, NJ, USA).
Propidium iodide solution
Manufactured by Keygen Biotech
Sourced in China
Propidium iodide solution is a fluorescent dye used in various cell biology applications. It is a nucleic acid intercalator that binds to DNA, allowing for the identification and quantification of cells with compromised cell membranes.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 fitc, by Keygen Biotech (3 mentions)
Annexin 5 fluorescein isothiocyanate fitc, by Keygen Biotech (1 mentions)
Facscalibur, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Coulter epics xl mcl system, by Beckman Coulter (1 mentions)
Caspase 3 activity kit, by Beyotime (1 mentions)
Bicinchoninic acid bca kit, by Beyotime (1 mentions)
Facsdiva software version 6, by BD (1 mentions)
Annexin 5 and propidium iodide, by Keygen Biotech (1 mentions)
Facscalibur flow cytometer, by Keygen Biotech (1 mentions)
Flow cytometry, by BD (1 mentions)
Annexin 5 fitc pi apoptosis assay kit, by Neobioscience (1 mentions)
Facscalibur analyzer, by BD (1 mentions)
9 protocols using propidium iodide solution
1
Apoptosis and Cell Cycle Evaluation
2
Cell Cycle and Apoptosis Analysis
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For the cell cycle experiment, cells were harvested and fixed with 70% ethanol. The fixed cells were centrifuged and washed thrice with phosphate-buffered saline (PBS) and then stained with propidium iodide (PI) solution (KeyGen Biotech Co. Ltd., Nanjing, China). For cell apoptosis assay, the cells were collected and washed twice with PBS. Annexin V-fluorescein isothiocyanate (FITC) (KeyGen Biotech Co. Ltd., Nanjing, China) and PI were added to the cell suspension, according to the manufacturer’s instructions. Cells in the fourth quadrant of the flow cytometry cytogram were regarded as apoptotic cells. All the samples were analyzed using a BD flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Each experiment was performed in triplicate.
3
Annexin V-FITC Apoptosis Assay
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Cells were collected, washed with phosphate-buffered saline (PBS), and resuspended in buffer at a concentration of 2 × 106 cells/ml. Cells were double-stained with Annexin V-FITC and propidium iodide solution (KEYGEN, Nanjing, China) and then examined by flow cytometry 10 min later.
4
Cell Cycle Analysis by Flow Cytometry
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The cells were harvested by trypsin digestion 48 h after transfection and washed in cold PBS before fixation in cold 75% alcohol at 4 °C overnight. Alcohol was removed and cells were again washed with cold PBS. Cells were stained with propidium iodide solution (KeyGEN BioTECH, China) containing 20 μg/ml RNase, and incubated at room temperature for 30 min. A FACS Calibur (BD,USA) flow cytometer was used to analyze the cell population. After filtration by a nylon mesh filter, cell cycle analysis was performed on a fluorescence-activated cell sorter (FACS, FACSVerse). Data were analyzed using FlowJo software (Version 7.6.1, Tree Star Software, San Carlos, CA, USA).
5
Cell Cycle Analysis of SGC-7901 Cells
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The SGC-7901 cells were seeded and cultured in 6-well plates (3×105 cells/well) for 24 h, and were then treated with 0.1% DMSO or ailanthone (1–4 µM) for 48 h at 37°C. Cells were harvested, washed with PBS and were fixed in 70% ethanol at 4°C overnight. Subsequently, cells were incubated with 1% RNase A at 37°C for 30 min and with propidium iodide solution (cat no. KGA511; Nanjing KeyGen Biotech Co., Ltd.) at 4°C for 30 min in the dark. The DNA content of cells was measured using a FACSCalibur flow cytometer (BD Biosciences). The experiment was independently repeated three times, and data were analyzed using the MultiCycle DNA content and cell cycle analysis software (FlowJo, version 7.6.5; FlowJo LLC, Ashland, OR, USA).
6
Apoptosis and Caspase-3 Assay Protocol
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After transfection for 2 days, cells were harvested, washed with PBS, and then stained with 5 μl Annexin V-FITC and 5 μl propidium iodide solution (KeyGen Biotech Co., Ltd.) for 15 min in the dark. Subsequently, the apoptosis rate was analyzed by flow cytometry (Coulter Epics XL-MCL system; Beckman Coulter, Inc., Brea, CA, USA).
For caspase-3 activity detection, the protein was extracted from the cells after transfection for 2 days using radioimmuno-precipitation assay (RIPA) lysis buffer and quantified with a bicinchoninic acid (BCA) kit (both from Beyotime Institute of Biotechnology, Shanghai, China). Subsequently, 5 μg protein was used to measure the caspase-3 activity using a caspase-3 activity kit (Beyotime Institute of Biotechnology). The OD was then measured using a plate reader (Bio-Rad Laboratories, Inc.) at 405 nm.
For caspase-3 activity detection, the protein was extracted from the cells after transfection for 2 days using radioimmuno-precipitation assay (RIPA) lysis buffer and quantified with a bicinchoninic acid (BCA) kit (both from Beyotime Institute of Biotechnology, Shanghai, China). Subsequently, 5 μg protein was used to measure the caspase-3 activity using a caspase-3 activity kit (Beyotime Institute of Biotechnology). The OD was then measured using a plate reader (Bio-Rad Laboratories, Inc.) at 405 nm.
7
Apoptosis Assay of Breast Cancer Cells
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Flow cytometry was used to detect the apoptosis of MCF-7 and MDA-MB-231 cells following transfection with different plasmids. Cells in the logarithmic growth phase were washed three times with PBS, harvested by using 0.25% trypsin, collected and centrifuged for 5 min (1,000 × g, at 4°C). MCF-7 and MDA-MB-231 cells were washed twice with PBS and centrifuged for 5 min (1,000 × g; 4°C), and then incubated with 5 µl Annexin V-FITC (cat. no. KGA108; Nanjing KeyGen Biotech Co., Ltd.) and 10 µl propidium iodide solution (cat. no. C1062M; Beyotime Institute of Biotechnology) for 20 min at room temperature in the dark. Cell apoptosis was detected by flow cytometry (cat. no. 322457; Bio-Rad Laboratories, Inc.) and data analysis was performed using FACSdiva software version 6.1.2 (BD Biosciences).
8
Cell Cycle and Apoptosis Assay Protocol
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For cell cycle assays, transfected cells were harvested, diluted to a density of 1 × 106 cells/mL and fixed with 70% ice-cold ethanol. Next, the cells were stained with 400 μL of a propidium iodide solution (Keygen Biotech, Nanjing, China) for 30 min and then subjected to cell cycle analysis using flow cytometry (BD Biosciences, San Jose, CA, USA).
For apoptosis analysis, 300 nM H2O2 was applied to induce apoptosis. Cells were collected 48 h after transfection and resuspended in binding buffer. The cells were then incubated with annexin V and propidium iodide (Keygen Biotech, Nanjing, China) for 15 min in the dark and then analyzed by flow cytometry on a FACSCalibur flow cytometer.
For apoptosis analysis, 300 nM H2O2 was applied to induce apoptosis. Cells were collected 48 h after transfection and resuspended in binding buffer. The cells were then incubated with annexin V and propidium iodide (Keygen Biotech, Nanjing, China) for 15 min in the dark and then analyzed by flow cytometry on a FACSCalibur flow cytometer.
9
Apoptosis and Cell Cycle Analysis of MDA-MB-231 Cells
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MDA-MB-231 cell apoptosis was analyzed using an Annexin V-FITC/PI apoptosis assay kit (Neobioscience, China). Briefly, cells grown in the DECM scaffolds were harvested with trypsin. Cells were then resuspended in binding buffer and stained with Annexin V-FITC and PI for 15 min at room temperature in the dark. For cell cycle analysis, MDA-MB-231 cells prepared from either 3D cultures (at Day 10) or 2D cultures were treated with cold 75% ethanol at 4 °C overnight. Fixed cells were washed with cooled PBS and incubated with propidium iodide solution (Keygen, China) for 30 min at room temperature. All analyses were performed using a FACS Calibur analyzer (BD Biosciences, USA) with FlowJo software (Tree Star Software, San Carlos, California, USA).
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