Detection of the Effect of Compound Staurosporine (STSP) on HeLa Cell Apoptosis Using Caspase 3/7 Apoptosis Detection Reagent: HeLa cells at a density of 4×10⁵ cells/mL were seeded into a 96-well white microplate with a transparent bottom, at a volume of 100 µL per well. After 24 hours, STSP (3-fold serial dilution) was added, and the cells were incubated at 37°C in a 5% CO₂ atmosphere for 6 hours. Subsequently, the Caspase 3/7 activity was detected in accordance with the instructions of the Caspase 3/7 Apoptosis Detection Reagent. Figure 1 shows the dose-response curve of fluorescence intensity measured between 30 minutes and 130 minutes after adding the detection reagent, and the table presents the calculated EC₅₀ value.
UA-Glo® Caspase 3/7 Assay
UA-Glo® Caspase 3/7 Assay
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Product Details
Product Details
Product Specification
| Synonyms | Caspase 3/7 Apoptosis Detection Kit |
| Stability & Storage |
Store at -20°C or below, protected from light. The shelf life is 12 months. |
Background
The ready-to-use Caspase 3/7 Apoptosis Detection Kit is designed for the quantitative measurement of Caspase 3 and 7 activity in cells. When the reagent is added to the target cells, Caspase 3/7 released from the lysed cells cleaves the Caspase 3/7-specific peptide-fluorophore conjugate substrate in the reagent, thereby releasing fluorophore. The luciferase in the reagent can catalyze the reaction of fluorophore to produce light, and the intensity of the light is proportional to the activity of Caspase 3/7.
This reagent features high signal-to-noise ratio, good repeatability and excellent stability. Its homogeneous ready-to-use formula reduces experimental preparation and operation steps, minimizing errors caused by multiple sample additions. Additionally, its stable glow signal makes the product particularly suitable for high-throughput compound screening.
Components
Luciferase, peptide-fluorophore conjugate substrate, and buffer are mixed and then filled into 10 ml or 100 ml brown bottles. The specifications are as follows:
|
Specification |
Number of detectable wells in a 96-well plate |
Number of detectable wells in a 384-well plate |
|
2.5 ml |
50 wells |
250 wells |
|
10 ml |
200 wells |
1000 wells |
|
100 ml |
2,000 wells |
10,000 wells |
|
10X10 ml |
2,000 wells |
10,000 wells |
Protocol
Cell Preparation
Seed cells at an appropriate density in a 96-well or 384-well white cell culture plate.
According to the experimental design, after incubating the cells for a certain period, add the test compound at an appropriate concentration to the wells of the cell plate. It is recommended to set up a cell control with only solvent vehicle (negative control) and a cell culture medium control with only solvent vehicle (blank control) (see Note 4 for details).
Continue culturing for an appropriate time to induce cell apoptosis as required by the experiment.
Caspase 3/7 Apoptosis Detection
Take out the Caspase 3/7 Apoptosis Detection Reagent and equilibrate at room temperature for 20 minutes. Mix gently by swirling.
Take out the cell culture plate to be tested and equilibrate at room temperature for 20 minutes.
Add 50 µl of the detection reagent to 100 µl of cells in a 96-well plate, or 10 µl of the reagent to 20 µl of cells in a 384-well plate. Shake the plate for 2 minutes, then incubate in the dark at room temperature for 30-60 minutes. The fluorescence signal generally reaches its maximum approximately 1 hour after adding the detection reagent. Plate reading can be performed as early as 30 minutes after reagent addition, but no later than 3 hours.
Read the fluorescence signal using a fluorescence microplate reader.
Guidelines
1.Dispense and store the reaction reagents according to the instruction manual to ensure reagent stability.
2.Without strict verification, it is not recommended to arbitrarily change the dosage of reaction reagents.
3.The luciferase reaction is sensitive to temperature changes. Reagents and test samples need to be equilibrated to room temperature (22℃-26℃), and the temperature should be kept constant (±1℃) during the test.
4.The blank control is used to measure the background fluorescence related to the cell culture system and Caspase 3/7 detection reagent. You can choose to subtract the blank control from the detection value before performing the corresponding calculation. The cell negative control reflects the basal Caspase 3/7 activity of the test cells and can be used as a reference for interpreting experimental results.
5.This product is for research use only.
Picture
Picture
Bioactivity
Sensitivity Test of Caspase 3/7 Apoptosis Detection Reagent: After treating HeLa cells with 1 μM STSP for 6 hours, the STSP-treated HeLa cells (2-fold serial dilution) were seeded into a 96-well white microplate with a transparent bottom at a volume of 100 μL per well. The cell concentration ranged from 25,000 to 180 cells per well. Caspase 3/7 activity was detected in accordance with the instructions of the Caspase 3/7 Apoptosis Detection Reagent. The left graph shows that the fluorescence signal was linearly correlated with the number of apoptotic cells (R² > 0.99) during the 40-minute to 70-minute detection period, and as few as 200 apoptotic cells could be detected. The right graph shows the results of Caspase 3/7 activity detection (fluorescence intensity measured at 70 minutes) after treating HeLa cells with 1 μM STSP and solvent DMSO for 6 hours.
