Cell apoptosis was examined with flow cytometry (DxFLEX; Beckman Coulter, Suzhou, Jiangsu, China) following annexin V/propidium iodide (PI) double staining (CA1020; Solarbio, Beijing, China). After the indicated treatment, the cells were trypsinized and rinsed with PBS and then resuspended in 100 μL of binding buffer. Five microliters of annexin V and 5 μL of PI were sequentially added and incubated for 5 min in the dark at room temperature. Subsequently, cells were diluted with 400 μL binding buffer, and at least 104 cells were analyzed in each treatment. Annexin V-positive cells were regarded as apoptotic cells. Therefore, cells in quadrants 1 and 4 of the flow cytometry dot plots with quadrant markers were considered apoptotic. Apoptosis rate = (annexin V positive cell number/total cell number) × 100%.
Ca1020
Manufactured by Solarbio
Sourced in China
The CA1020 is a centrifuge designed for general laboratory use. It features a sturdy construction and a digital display for setting the desired speed and time. The centrifuge can accommodate a variety of rotor sizes and sample volumes.
Automatically generated - may contain errors
Lab products found in correlation
Dxflex, by Beckman Coulter (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
C0080, by Solarbio (1 mentions)
Lsr 2, by BD (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Flow cytometry, by BD (1 mentions)
Annexin 5 fitc pi, by Solarbio (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Solarbio (1 mentions)
Mod t lt software, by Verity Software House (1 mentions)
Facs caliber cytometer, by BD (1 mentions)
8 protocols using ca1020
1
Flow Cytometry Analysis of Cell Apoptosis
2
Annexin V-FITC Apoptosis Assay
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Cells in the different groups were cultured in a 6-well plate at a density of 2×104 cells/well for 24 h. The cells were then centrifuged, collected and washed with PBS twice. The supernatant was discarded, and the cells were resuspended in 400 µl of 1X Binding Buffer and incubated with 5 µl of Annexin V-FITC for 15 min in the dark. Cells were mixed thoroughly with 10 µl of PI staining solution (CA1020, Beijing Solarbio Science & Technology Co., Ltd.) and incubated for 5 min in the dark. The proportion of cell apoptosis was assessed using a flow cytometer (CytoFLEX, Beckman Coulter, Inc.).
3
Cell Cycle and Apoptosis Analysis
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The density of trypsinized cells was adjusted to 3 × 105 cells/mL, which were seeded in 6-well plates and cultured for 48 h. The single cell suspension was centrifuged at 1000 r.min−1 × 5 min followed by removal of the supernatant. Cells were fixed with 70% ice ethanol solution at − 20 °C or 1 h and then added with 20 μL RNA enzyme and reacted at 37 °C for 30 min. Subsequently, cells were incubated with 400 μL propidium iodide (PI) (C0080, Solarbio) for 15 min in the dark, on ice. The cell cycle was detected by a flow cytometer (BDLSR II, BD, FL, NJ, USA) with the excitation light wavelength at 488 nm. For detection of cell apoptosis, the cells were resuspended with 400 μL of Annexin V binding solution (CA1020, Solarbio) to 3 × 105 cells/mL and then incubated with 50 μL Annexin V-Fluorescein Isothiocyanate staining solution on ice, in the dark, for 15 min. After the addition of 10 μL PI staining solution, cell apoptosis was detected by flow cytometer also with the excitation light wavelength as 488 nm.
4
Apoptosis and Cell Cycle Analysis
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Cell samples were harvested using trypsin and washed with PBS for three times. Apoptosis and cell cycle distribution were detected through flow cytometry analysis of Annexin V staining and PI staining (Solarbio, CA1020) according to the standard routine. Living (Annexin V−/PI−), early apoptotic (Annexin V+/PI−), late apoptotic (Annexin V+/PI+), and necrotic (Annexin V−/PI+) cells were distinguished. Data were analyzed by FlowJo.
5
Quantifying Apoptosis and Necrosis
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To assess apoptotic cells, the Accuri C6 flow cytometer (BD, USA) was employed, following labeling with Annexin V-FITC and propidium iodide (PI) (CA1020; Solarbio, China). Cells were harvested, washed with PBS, and suspended in a binding buffer for Annexin V-FITC and PI staining, as per the manufacturer's instructions. After incubation, the labeled cell suspension underwent flow cytometry analysis using appropriate laser and filter settings to detect Annexin V-FITC and PI fluorescence. Data were analyzed using FlowJo software to quantify apoptotic and necrotic cell populations.
6
Cytotoxicity assay of compound 7f
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Treated A549 cells and PC-9 cells with 0 μM, 0.5 μM, 1.0 μM, or 1.5 μM 7f for 24 h; then, according to the operating instructions of the kit (CA1020, Solarbio, China), the cells were stained by PI (Solarbio, China) or Annexin V-FITC/PI (Solarbio, China) and analyzed by flow cytometry (Becton Dickinson and Company, USA).
7
Annexin V-FITC/PI Apoptosis Assay
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The following experiments were carried out according to the instructions of the Annexin V‐FITC/PI Apoptosis Detection Kit (CA1020; Solarbio). The cells were inoculated into six‐well plates (5 × 105 cells/well). Different groups of cells were pretreated according to experimental conditions. After discarding the medium, trypsinize the cells with EDTA‐free trypsin, add cell culture medium to terminate the digestion, and collect the digested cells into a centrifuge tube. The cells were centrifuged at 106 g for 5 min, and the supernatant was discarded. Add 1 mL of 4 °C pre‐chilled 1 × PBS to resuspend the cells, and repeat the centrifugation step once. Dilute the Binding Buffer with deionized water at a 1 : 9 dilution ratio. Resuspend cells in 1 × binding buffer to maintain a cell concentration of 1–5 × 106 cells·mL−1. Then take 100 μL of cell suspension and 5 μL Annexin V/FITC solution, mix well, and incubate at room temperature for 5 min in the dark. Finally, 5 μL of propidium iodide solution (PI, CA1020; Solarbio) and 400 μL of PBS were added and mixed, and flow cytometry was performed immediately.
8
Cell Cycle and Apoptosis Analysis of U2OS Cells
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Cell cycle of U2OS cells was examined with propidium iodide (PI) kit (CA1510, Solarbio). Transfected U2OS cells (5 × 10 4 /dish) were cultured in 6 cm dishes. After 72 h culture, cells were xated in 70% ethanol overnight at 4°C, washed by PBS and suspended in 100 µL R RNase A solution. Next, after incubation at 37 °C for 30 min, cells were treated with 400 µL PI for 30 min at 4 °C. Cell populations in in each of the cell cycle phases were quanti ed using FACS Caliber cytometer (BD Biosciences) and the data were analyzed by the Mod t LT software (Verity Software House, USA).
For apoptosis experiment, transfected U2OS cells were harvested and dyed by both Annexin V-FITC and PI as manufacturers' protocol (CA1020, Solarbio). Brie y, harvested U2OS cells were centrifuged at 300× g for 10 min. After discarding the supernatant, the cells were resuspended in 1 ml 1 × binding buffer and the cell concentration was adjusted to 1×10 6 cells/ml. 100 µL of cells (1 × 10 5 ) was added to each labelled tube, incubated with 5 µL Annexin V-FITC for 10 min and then incubated with 5 µL PI for 5 min.
For apoptosis experiment, transfected U2OS cells were harvested and dyed by both Annexin V-FITC and PI as manufacturers' protocol (CA1020, Solarbio). Brie y, harvested U2OS cells were centrifuged at 300× g for 10 min. After discarding the supernatant, the cells were resuspended in 1 ml 1 × binding buffer and the cell concentration was adjusted to 1×10 6 cells/ml. 100 µL of cells (1 × 10 5 ) was added to each labelled tube, incubated with 5 µL Annexin V-FITC for 10 min and then incubated with 5 µL PI for 5 min.
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