No More Sales-Annexin V-Flamma® 488 Apoptosis detection kit
Product#: Inactivated-XAP1201
$99,999.00
Packing Unit
-
50 tests
-
250 tests
Availability:
Ships in 1-2 Weeks
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Annexin V-Flamma® 488 Apoptosis detection kit
Cat. No. List below
Description
Annexin V-Flamma® 488 apoptosis detection kit, consisting of Flamma 488-conjugated annexin V, propidium iodide (PI) and the binding buffer, is a rapid and selective detection kit for labeling of externalized PS, an indicator of intermediate stages of apoptosis. Flamma® dyes display strong absorption, high fluorescence quantum yield and high photostability, and they maintain good fluorescence activity and stability after conjugation to biomolecules. The maxima of Ex/Em are at 495/519 nm. Annexin V-Flamma® 488 kit can be utilized in fluorescent imaging as well as for flow cytometry of apoptotic cells. This efficient apoptosis detection kit allows to accurately analyze mixed populations of apoptotic and normal cells.
Specifications
- Fluorophore: Flamma® 488
- Application: Apoptotic cell imaging
- Excitation/Emission Max.(nm): 495/519 nm
- Appearance: Liquid
- Components: Annexin V-FITC, PI, binding buffer
- Storage conditions: 4 ℃, protect from light
Table 1. List of Annexin V-Flamma® apoptosis detection kits
| Quick link (Cat.#) | Series | λEx (nm) | λEm (nm) |
| XAP1102 | Annexin V-FITC Apoptosis detection kit | 492 | 519 |
| XAP1201 | Annexin V-Flamma® 488 Apoptosis detection kit | 549 | 564 |
| XAP1301 | Annexin V-Flamma® 552 Apoptosis detection kit | 648 | 672 |
| XAP1801 | Annexin V-TAMRA Apoptosis detection kit | 648 | 672 |
| XAP2102 | Annexin V-FITC | 492 | 519 |
| XAP2202 | Annexin V-Flamma® 488 | 495 | 519 |
| XAP2302 | Annexin V-Flamma® 552 | 550 | 565 |
| XAP2802 | Annexin V-TAMRA | 543 | 575 |
| XAP2402 | Annexin V-Flamma® 648 | 648 | 663 |
| XAP2022 | Annexin V-Flamma® 675 | 675 | 691 |
| XAP2032 | Annexin V-Flamma® 749 | 749 | 774 |
| XAP2042 | Annexin V-Flamma® 774 | 774 | 806 |
| XAP2052 | Annexin V-ICG | 785 | 821 |
Background
Annexin V-Flamma® apoptosis detection kit consists of Flamma® conjugated Annexin V, Propidium iodide (PI) and Aannexin V binding buffer. Phosphatidyl serine (PS) is located on the cytoplasmic surface of the cell membrane, however when apoptosis is in progress, PS is translocated to the outer leaflet of the plasma membrane exposing to the external cellular environment. Annexin V is a Ca2+ dependent PS-binding protein that being used to detect PS on the cell surface. PI is impermeable to live cells and the early stage apoptotic cells, but it binds to the nucleic acids of dead cells and emits red fluorescence. Normal cells are not labeled by either Annexin V or PI, early stage apoptotic cell is stained with Annexin V and natural death or cell subject to necrosis is stained with PI. The late stage apoptotic cells are stained by both reagents. This kit allows to identify apoptotic cells using fluorescence microscopic or flow cytometry analysis with simple staining procedures. Sample analysis is performed on live cells and does not require cell fixation process.
Annexin V-Flamma® apoptosis detection kit consists of Flamma® conjugated Annexin V, Propidium iodide (PI) and Aannexin V binding buffer. Phosphatidyl serine (PS) is located on the cytoplasmic surface of the cell membrane, however when apoptosis is in progress, PS is translocated to the outer leaflet of the plasma membrane exposing to the external cellular environment. Annexin V is a Ca2+ dependent PS-binding protein that being used to detect PS on the cell surface. PI is impermeable to live cells and the early stage apoptotic cells, but it binds to the nucleic acids of dead cells and emits red fluorescence. Normal cells are not labeled by either Annexin V or PI, early stage apoptotic cell is stained with Annexin V and natural death or cell subject to necrosis is stained with PI. The late stage apoptotic cells are stained by both reagents. This kit allows to identify apoptotic cells using fluorescence microscopic or flow cytometry analysis with simple staining procedures. Sample analysis is performed on live cells and does not require cell fixation process.
- Induce apoptosis in cells using the desired method. Prepare a negative control by incubating cells in the absence of inducing agent.
- After the incubation period, harvest cells, and wash the cells twice with cold PBS buffer.
- Prepare 1X Annexin V binding buffer. For example, for making 5 mL 1X buffer, dilute 1 mL 5X Annexin V binding buffer (Component C) with 4 mL deionized water.
- Prepare a 100 µg/mL PI working solution by diluting 5 µL of 1 mg/mL PI stock solution (Component B) with 45 µL 1X Annexin V binding buffer. Store the solution in the dark, and unused portion of PI working solution can be saved for future usage.
- Centrifuge the washed cells from step 2, discard the supernatant and suspend the cells in 1X Annexin V binding buffer. Determine the cell density and dilute in Annexin V binding buffer to ~ 1 × 106 cells/mL.
- Add 5 ~ 25 µL of Annexin V-Flamma® Flours (Component A) and 1 ~ 2 µL of the 100 µg/mL PI working solution (prepared in step 4) to each 100 µL of cell suspension. Higher concentrations of the Annexin V-Flamma® Flours tend to produce better results; however, the optimal staining concentration needs to be determined empirically. Incubate the cells at room temperature for 15 minutes.
- After the incubation period, add 400 µL 1X Annexin V binding buffer, mix gently and keep the samples on ice.
- Analyze the stained cells with flow cytometry. The population should separate into three groups: live cells show only a low level of fluorescence, apoptotic cells show corresponding Annexin V-Flamma® fluorescence, and dead cells show fluroescence of both PI and Annexin V-Flamma® Flours
| Annexin-V Flamma® Apoptosis | Fluorescence channel |
| Annexin V-FITC | FITC or 488 |
| Annexin V-Flamma® 488 Apoptosis | FITC or 488 |
| Annexin V-Flamma® 552 Apoptosis | TRITC or Cy3 |
| Annexin V-Flamma® 648 Apoptosis | Cy5 |
Adherent cells with fluorescence microscopy
- Prepare 1 x 105 cells/mL of cultured cells on the chamber slide. Induce apoptosis in cells using the desired method. Prepare a negative control by incubating cells in the absence of inducing agent.
- Incubate cells in at 37 ℃ for appropriate period, After the incubation, remove supernatant, and wash the cells twice with cold PBS.
- Prepare 1X Annexin V binding buffer. For example, to make 5 mL 1X buffer, add 1 mL 5X Annexin V binding buffer (Component C) to 4 mL deionized water.
- Prepare a 100 µg/mL working solution of PI by diluting 5 µL of 1 mg/mL PI stock solution (Component B) in 45 µL 1X Annexin V binding buffer. Store the solution in the dark, and unused portion of this working solution saved for future experiments.
- Re-suspend the washed cells from step 2 with 1X Annexin V binding buffer. Discard the supernatant and add 100 ~ 500 µL of 1X Annexin V binding buffer to each well.
- Add 5 ~ 25 µL of Annexin V-Flamma® Flours (Component A) and 1 ~ 2 µL of the 100 µg/mL PI working solution (prepared in step 4) to each well. Higher concentrations of the Annexin V conjugate tend to produce better results; however, the optimal staining concentration needs to be determined empirically. Incubate the cells at room temperature for 15 minutes in the dark.
- After the incubation period, wash the cells with 1X Annexin V binding buffer, add 500 µL 1X Annexin V binding buffer to each well and cover the well with coverslip.
- Analyze the stained cells using fluorescence microscope. Observe the fluorescence using filters for PI and appropriate Annexin V-Flamma® fluorescence channel. The cells should separate into three groups: live, apoptotic, and dead. Live cells show only weak Annexin V staining of the cellular membrane, while apoptotic cells show a significantly higher degree of surface labeling. Dead cells show both membrane staining by Annexin V and strong nuclear staining from the propidium iodide.
Citation & Reference
1. Zeng, Wenbin. Molecular Imaging of Apoptosis: From Micro to Macro. Theranostics 5.6 (2015): 559
