4. A405 of each standard was subtracted from A405 of the blank control without pNA to calculate the actual absorbance due to pNA, and a standard curve of pNA concentration versus A405 was created. The pNA standard curve has a good linear relationship in the range of 0-20uM, as shown in the figure below.Note: The experimental data may vary due to different experimental conditions, testing instruments, etc. The data in the figure is for reference only.

3. Sample collection
1. Suspended cells: Centrifuge the control sample that did not induce apoptosis and the sample that induced apoptosis at 600g at 4 º C for 5 minutes to collect the cells, carefully aspirate the supernatant, and ensure that no cells are aspirated as much as possible, and wash once with PBS. After the supernatant was aspirated as before, the lysate was added at a ratio of 100 ul of lysate per 2 million cells, the pellet was resuspended, and lysed in an ice bath for 15 minutes. Go down to Step 4.
2. Adherent cells: suck the cell culture medium for later use. The adherent cells were digested with trypsin and collected into a spare cell culture medium. Collect cells by centrifugation at 600 g at 4 º C for 5 minutes, carefully aspirate the supernatant while ensuring that no cells are aspirated as much as possible, and wash once with PBS. After the supernatant was aspirated as before, the lysate was added at a ratio of 100 ul of lysate per 2 million cells, the pellet was resuspended, and lysed in an ice bath for 15 minutes. Go down to Step 4.
3. Tissue samples: Add lysate at a ratio of 100ul lysate per 3-10mg tissue, and homogenize with a glass homogenizer on an ice bath. The homogenate was then transferred to a 1.5 ml centrifuge tube and lysed in an ice bath for an additional 5 minutes.
4. Centrifuge at 4 º C 16,000-20,000 g for 10-15 minutes.
5. Transfer the supernatant to an ice bath precooled centrifuge tube.
6. The enzyme activity of Caspase 1 was determined immediately or the sample was stored at − 80 ° C. At the same time, a small amount of sample can be taken to measure the protein concentration by Bradford method, and try to make the protein concentration reach 1-3mg/ml, which is equivalent to at least 10-30ug of protein per 10ul of the sample to be tested. If there are fewer cells, the amount of cells can be appropriately increased.

4. Detection of Caspase 1 enzyme activity
1. Take out an appropriate amount of Ac-YVAD-pNA (2mM) and place it on an ice bath for later use.
2. Set up the reaction system as follows:

	Blank control	Sample
Assay buffer	40ul	40ul
Sample to be measured	0ul	50ul
Lysate	50ul	0ul
Ac-YVAD-pNA (2 mM)	10ul	10ul
Total Volume	100ul	100ul

Note: When setting the reaction system, add the detection buffer first, then add the sample to be tested, and mix properly. Be careful to avoid bubbles during mixing. An additional 10 ul of Ac-YVAD-pNA (2 mM) was then added.

3. Add Ac-YVAD-pNA (2mM) and mix well, taking care to avoid bubbles during mixing. Incubate at 37 º C for 60-120 minutes. A405 can be measured when the color change is obvious. If the color change is not obvious, the incubation time can be appropriately extended, or even overnight.
4. A405 of the sample minus A405 of the blank control is the absorbance produced by pNA catalyzed by Caspase 1 in the sample. The amount of pNA catalytically produced in the sample can be calculated by comparing it with the standard curve obtained in step 2.
5. Refer to the definition of Caspase 1 enzyme activity unit from Chemicon: One unit is the amount of enzyme that will clear 1.0 nmol of the colorimetric substrate Ac-YVAD-pNA per hour at 37 º C under saturated substrate concentrations. That is, an enzyme activity unit is defined as the amount of caspase 1 enzyme that can cleave 1 nmol of Ac-YVAD-pNA to produce 1 nmol of pNA within one hour at 37 º C when the substrate is saturated. In this way, it is possible to calculate how many enzyme activity units of Caspase 1 are contained in the sample. Note: In the detection system of this kit, the initial concentration of the substrate is 0.2 mM, at which time the substrate is saturated. For many samples, the substrate is saturated within 2 hours of incubation at 37 º C; If the activity of caspase 1 enzyme in the sample is particularly high, the sample must be properly diluted with lysate before determination.
6. Use the Bradford method to detect the protein concentration in the sample to be tested (because the lysate contains a high concentration of DTT, it is not suitable to use the BCA method for protein concentration determination). In this way, the enzyme activity unit of Caspase 1 contained per unit weight of protein in a sample can be calculated.