| Product Description |
Superview ™ 488 caspase-3 substrate provides an effective tool for detecting apoptosis based on caspase-3/7 viability, which is suitable for fluorescence microscopy and flow cytometry. Compared with other fluorescent substrates or fluorescent inhibitors of caspases based on (Flica) analysis, superview ™ 488 caspase-3 substrate does not inhibit the apoptosis process of intact cells while detecting caspase-3/7 activity. Substrate consists of a fluorescent DNA dye that couples the caspase-3/7 devd recognition sequence. Substrate is initially nonfluorescent and crosses the cell membrane into the cytoplasm. In apoptotic cells, caspase-3/7 cleaves substrate and releases high affinity DNA staining, which migrates to the nucleus to label DNA and emits bright green fluorescence. Therefore, superview ™ 488 caspase-3 substrate is bifunctional, which can not only detect caspase-3/7 activity, but also visualize the morphological changes of the nucleus during apoptosis. Superview ™ 488 staining can be formaldehyde fixed and compatible with subsequent immunostaining experiments. Superview ™ 488 caspase-3 substrate provides two dissolved forms, DMSO and PBS. The PBS form can be used for cells sensitive to DMSO toxicity, and in cell types insensitive to DMSO, the addition of DMSO dissolved form can enhance, superview ™ 488 incubation staining effect. Product parameters： aladdin 488：Ex/Em = 500/530 nm (with DNA)
Matters needing attention： 1. cells can be treated with a final concentration of 1 μ M Hoechst 33342 dye CO staining, resulting in blue fluorescent staining of the nucleus (excitation / emission: 346 / 460 nm). 2. superview ™ 488 staining can be fixed by formaldehyde, but is incompatible with methanol fixation. 3. formaldehyde fixed superview ™ 488 stained cells can be treated with 0.1% Triton X-100 for subsequent staining, but the brightness of the staining after such treatment may be weakened. Scope of application： Caspase-3 substrate, enzyme substrate Usage:
1. Experimental optimization
The experimental steps provided below are based on the endpoint detection system. Aladdin 488 Substrate can also be used for long-term cell incubation studies. Cell density, substrate concentration, and inhibitor concentration may need to be optimized. The optimal substrate concentration may be between 1-10 μ Between M. Cells can be incubated with substrates in culture medium, PBS, or other buffer of your choice. For adherent cells, we recommend replacing them with fresh culture media containing substrates to prevent background heterogeneity. The operation of changing the medium or washing the cells after substrate incubation is freely selectable. When used in cell types that are not sensitive to DMSO, adding DMSO may enhance the staining effect of Aladdin 488.
2. We suggest that you set the following controls:
A. Negative control: cells that do not induce apoptosis
B. Positive control: Cells that induce apoptosis
3. Flow cytometry
(1) Choose appropriate methods to induce cell apoptosis, with untreated cell samples as controls.
(2) Adhering cells for SuperView ™ Before conducting the 488 Caspase-3 experiment, the cells were digested using trypsin or other methods.
(3) Resuspend cells with culture medium or buffer to achieve a cell density of 106/mL.
(4) Suck 0.2 mL of cell suspension into a flow cytometry test tube.
(5) To 200 μ Add 1 to L cell suspension μ Substrate of L 1 mM and immediately mix to achieve a substrate concentration of 5 μ M. The optimal substrate concentration for different cells may vary and requires analysis and optimization.
(6) Incubate cells at room temperature in dark for 15-30 minutes.
(7) Join 300 μ L-medium or PBS, analyzed by flow cytometry. Channel for detecting green fluorescence (excitation/emission: 485/515 nm).
4. Fluorescence microscope
(1) Choose appropriate methods to induce cell apoptosis, with untreated cell samples as controls. (2) Using a solution containing 5 μ Fresh culture medium or PBS from M Substrate was used to replace the cells (see experimental optimization).
(3) Incubate cells at room temperature for 30 minutes or longer.
(4) Cells can be directly observed in culture media containing Substrate. For endpoint analysis, PBS is used to clean cells, fluorescence microscopy is used to observe cells, and a filter (excitation/emission: 485/515 nm) is used to observe green fluorescence.
5. Fluorescence enzyme-linked immunosorbent assay (ELISA) reader
(1) Cultivate adherent cells in black 96 well plates; Suspend cells, adjust the density to 106 cells/mL, and divide 0.2 mL of cell suspension into one well. (2) Choose appropriate methods to induce cell apoptosis, with untreated cell samples as controls. Note: Cells can be processed in tubes or bottles and then transferred to a 96 well detection plate. (4) Incubate cells at room temperature in dark for 15-30 minutes.
(5) For suspended cells, gently shake to resuspend the cells. The fluorescence enzyme-linked immunosorbent assay instrument is set with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. Suggest using bottom collection method for adherent cells. Changes in the density of adherent cells may lead to inaccurate readings. Frequently asked question
| Problem |
Answer |
| How stable is it? |
The stability of this substance is very good, and users have reported that the product has been left at 37 ℃ for 4-5 days, but the effect is still very good |
| When will it be added to the cell? |
This substance can be added to cells in the early and later stages of the experiment, and it does not affect the process of cell apoptosis, which can be monitored in real-time Caspase-3 activity. |
| Can it be used for tissue staining? |
Our company has not conducted live tissue staining experiments, and there are literature reports that it can be used for embryonic tissue or three-dimensional cell culture. |
| Can it be used for subsequent immunostaining? |
Sure. It is recommended to use 2-4% paraformaldehyde to fix at room temperature for 10-15 minutes, as prolonged fixation time can lead to signal degradation. |
| How specific is it? |
Similar to other caspase-3 substrates, Caspase-3 Substrate is based on the ability to be cleaved by Caspase-7-cleaved DEVD caspase-3 common sequence, other caspases may also be associated with substrate specific sequences
Splitting DEVD substrates due to column overlap |
| Applicable to which cells? |
Caspase-3 Substrate has been reported to be used for immortality in various primary cells and scientific literature in biochemical cells |
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F2420392