For each group, cells were collected and washed twice with PBS. After fixation with 70% glacial ethanol at 4°C overnight, cells were centrifuged and washed three times with cold PBS, RNase A solution (100 µL) was added, and cells were incubated for 30 minutes at 37°C. A PI staining solution (KeyGen Biotech, Nanjing, China) was added to give a final concentration of 400 µL, and then, the solution was mixed uniformly. After 30 minutes staining at 4°C in the dark, fluorescence detection was performed by flow cytometry. Experiments were repeated three times.
Pi staining solution
Manufactured by Keygen Biotech
Sourced in China
The PI staining solution is a laboratory reagent used for the detection and quantification of DNA content in cells. It binds to the DNA and emits fluorescence, allowing for the analysis of cell cycle stages and DNA content using flow cytometry or fluorescence microscopy.
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5 protocols using pi staining solution
1
Cell Cycle Analysis by Flow Cytometry
2
Cell Cycle and Apoptosis Analysis
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Seventy-two hours after siRNA transfection, HEK-293T cells, HUVECs and LO2 cells were detached with 0.25% trypsin and washed twice with PBS, and the cell density was adjusted to 1 × 106 cells/ml. Next, the cells were fixed with precooled 70% alcohol overnight at 4 °C, washed twice with PBS, and treated with 500 µl of a propidium iodide (PI) (KeyGEN BioTECH, Nanjing, China) reaction mixture containing RNase in the dark for 60 min at room temperature. Cell cycle phases were detected by flow cytometry (FACS Calibur, Becton, Dickinson and Company, New Jersey, USA), and the data were analyzed by FlowJo software. For apoptosis detection, cells were collected and washed as described above, mixed with 500 µl binding buffer containing 5 µl Annexin V-FITC and 5 µl PI staining solution (KeyGEN BioTECH, Nanjing, China), and incubated in the dark for 15 min at room temperature. A flow cytometer was used to detect apoptosis, and the data were analyzed by FlowJo.
3
Quantifying Cell Apoptosis by Flow Cytometry
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Cell apoptosis was measured by flow cytometry assay. Briefly, the transfected H9C2 cells (1 × 106 cells) in each treatment group were collected and washed with PBS. Each precipitate was resuspended in 400 μL PBS. After that, the Annexin V-FITC (5 μL, Keygen, Nanjing, China) and PI staining solution (5 μL, Keygen, Nanjing, China) were added and incubated in dark for 30 min. At last, the cell apoptotic rate was examined by flow cytometry (FACSCalibur, BD Biosciences) and was analyzed by FlowJo software (Version 7.6.1).
4
Annexin V-FITC Apoptosis Assay
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Annexin V-Fluorescein Isothiocyanate Apoptosis Detection Kit (KeyGen Biotech, Nanjing, China) was used to detect cell apoptosis. The cells were digested by non-ethylene diamine tetra acetic acid (EDTA)-containing trypsin 48 hours after transfection and the treatment of lead, and then centrifuged (300 × g, 4°C, 5 min). Approximately 1-5 × 105 cells were collected and resuspended in 100 μl 1×Binding Buffer (KeyGen Biotech). Annex in V-FITC (5 μl KeyGen Biotech) and 5 μl PI Staining Solution (KeyGen Biotech) were added and mixed gently. The reaction lasted for 10 minutes under room-temperature in a light-proof environment. Four hundred micro liters of 1×Binding Buffer was added and mixed gently. FCM was performed within one hour using a flow cytometer (BD Biosciences, NJ, USA). Annexin V-FITC+PI- quadrant represented early apoptotic cells; Annexin V-FITC+PI- quadrant represented early apoptotic cells (LR); Annexin V-FITC-PI+ quadrant represented necrotic cells (UL); Annexin V-FITC+PI+ quadrant represented late apoptotic and necrotic cells (UR); Annexin V-FITC-PI- quadrant represented live cells (LL). Finally, the ratio of apoptotic cells accounting for total cells was calculated.
5
Annexin V-FITC and PI Apoptosis Assay
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The cells were collected, washed once with PBS, centrifuged at 1000 r/min for 5 minutes, and the supernatant was discarded. According to the kit instructions, each sample (293T‐WT, 293T‐KO, HepG2‐WT, HepG2‐KO) was resuspended in 100 μL binding buffer and then Annexin V‐FITC (5 μL/tube) and PI staining solution (5 μL/tube) (KeyGen, China) were added and incubated for 10 minutes in the dark. Next, 400 μL binding buffer was added to each tube, tubes were gently mixed, and flow cytometry analysis was performed within 1 hours.
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