Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Green Fluorescence Optimized for Flow Cytometry*
![The detection of binding activity of Annexin V-iFluor® 488 and phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Blue) or with 20 µM camptothecin (Red) in a 37 °C, 5% CO2 incubator for 4-5 hours, and then dye loaded with Annexin V-iFluor® 488 for 30 minutes. The fluorescence intensity of Annexin V-iFluor® 488 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using the FL1 channel.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-meter-annexin-v-binding-apoptosis-assay-kit-green-fluorescence-optimized-for-flow-cytometry%2Ffigure-for-cell-meter-annexin-v-binding-apoptosis-assay-kit-green-fluorescence-optimized-for-flow-cytometry_INjw1.jpg&w=640&q=75)
![The detection of binding activity of Annexin V-iFluor® 488 and phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Blue) or with 20 µM camptothecin (Red) in a 37 °C, 5% CO2 incubator for 4-5 hours, and then dye loaded with Annexin V-iFluor® 488 for 30 minutes. The fluorescence intensity of Annexin V-iFluor® 488 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using the FL1 channel.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-meter-annexin-v-binding-apoptosis-assay-kit-green-fluorescence-optimized-for-flow-cytometry%2Ffigure-for-cell-meter-annexin-v-binding-apoptosis-assay-kit-green-fluorescence-optimized-for-flow-cytometry_INjw1.jpg&w=640&q=75)
![The detection of binding activity of Annexin V-iFluor® 488 and phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Blue) or with 20 µM camptothecin (Red) in a 37 °C, 5% CO2 incubator for 4-5 hours, and then dye loaded with Annexin V-iFluor® 488 for 30 minutes. The fluorescence intensity of Annexin V-iFluor® 488 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using the FL1 channel.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-meter-annexin-v-binding-apoptosis-assay-kit-green-fluorescence-optimized-for-flow-cytometry%2Ffigure-for-cell-meter-annexin-v-binding-apoptosis-assay-kit-green-fluorescence-optimized-for-flow-cytometry_INjw1.jpg&w=128&q=25)
![Effect of FSS on apoptosis and necrosis in tubular cells. Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. A/ Cells were stained with Annexin-V and then immediately subjected to analysis of phosphatidylserine externalization (Annexin-V fluorescence, X-axis) and Propidium Iodure (PI) uptake (PI fluorescence, Y-axis) using flow cytometry. Living, early apoptotic or necrotic (primary or secondary) cells were distinguished by the criteria of Annexin-V−/PI−(bottom left quadrant), Annexin-V+/PI− (bottom right quadrant) and Annexin-V+/PI+ (upper right quadrant), respectively. B/ Proportions of early apoptosis and necrosis cells were quantified and results are expressed as a percentage of the total population of cells. Data represent mean ± SEM of 7 experiments. *HK-2 cells were assessed for apoptosis and necrosis using Cell Meter Annexin V Binding Apoptosis Assay Kit (AAT Bioquest), according to the manufacturer's instructions. Source: Graph from <strong>Shear Stress-Induced Alteration of Epithelial Organization in Human Renal Tubular Cells</strong> by Damien Maggiorani, et al., <em>PLoS ONE</em>, July 2015.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-meter-annexin-v-binding-apoptosis-assay-kit-green-fluorescence-optimized-for-flow-cytometry%2Ffigure-for-cell-meter-annexin-v-binding-apoptosis-assay-kit-green-fluorescence-optimized-for-flow-cytometry_yplww.png&w=128&q=25)
AT A GLANCE
Prepare cells with test compounds (200 µL/sample).
Add Annexin V-iFluor® 488 stock solution.
Incubate at room temperature for 30 - 60 minutes.
Analyze cells using a flow cytometer with a 530/30 nm filter (FITC channel).
Before starting the experiment, thaw the vial of 100X Propidium Iodide (Component C) at room temperature.
CELL PREPARATION
For guidelines on cell sample preparation, please visit:
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
Treat cells with test compounds for a desired period of time (4 - 6 hours for Jurkat cells treated with camptothecin) to induce apoptosis.
Note: Annexin V flow cytometric analysis on adherent cells is not routinely tested since specific membrane damage may occur during cell detachment or harvesting. However, methods for utilizing Annexin V for flow cytometry on adherent cell types have been previously reported by Casiola-Rosen et al. and van Engelend et al.
Centrifuge the cells to get 1 - 5 × 105 cells/tube.
Resuspend cells in 200 µL of Assay Buffer (Component B).
Add 2 µL of Annexin V-iFluor® 488 (Component A) into the cells.
Optional: Add 2 µL of 100X Propidium Iodide (Component C) for necrosis cells.
Incubate at room temperature for 30 to 60 minutes, protected from light.
Optional: Add 200 to 300 µL of Assay Buffer (Component B) to increase volume before analyzing the cells with a flow cytometer.
Monitor the fluorescence intensity of Annexin V-iFluor® 488 using a flow cytometer with a 530/30 nm filter (FITC channel). Measure the cell viability using the 610/20 nm filter (PE-Texas Red channel) after adding propidium iodide to the cells.
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Orange Fluorescence Optimized for Flow Cytometry* | 557 | 570 | 1000001 | 0.641 | 0.23 | 0.14 |
Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Red Fluorescence Optimized for Flow Cytometry* | 587 | 603 | 2000001 | 0.531 | 0.05 | 0.04 |
Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Deep Red Fluorescence Optimized for Flow Cytometry* | 656 | 670 | 2500001 | 0.251 | 0.03 | 0.03 |
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