A cell apoptosis detection kit and its detection method

Abstract

This invention provides a cell apoptosis detection kit and its detection method. The cell apoptosis detection kit comprises the following raw materials in the specified volumes: 2-3 μL annexin V, 0.5-1.5 mL binding buffer, 0.5-1.5 mL blocking buffer, 2-3 μL reactive dye I, and 4-6 μL reactive dye II. The binding buffer is composed of steroidal saponins, calcium ion solution, 4-hydroxyethylpiperazine ethanesulfonic acid, and water in a mass-to-volume ratio of (0.6-1.0):(0.05-0.1):(0.8-1.2):(96-98) g / mL. The blocking buffer is composed of protamine, sodium chloride solution, and tris(hydroxymethyl)aminomethane hydrochloride solution in a mass-to-volume ratio of (0.6-0.8):(6-8):(6-10) g / mL. The apoptosis detection kit and detection method prepared by this invention can reduce fluorescence quenching of active dyes, improve the binding ability of annexin V and phosphatidylserine, thereby improving the detected apoptosis rate and cell necrosis rate, and improving the accuracy of the detection results.

Description

Technical Field

[0001] This invention relates to the field of seed cell detection technology, specifically to a seed cell apoptosis detection kit and its detection method. Background Technology

[0002] Apoptosis, also known as programmed cell death, is a gene-controlled, autonomous, and orderly process of cell death. This process involves the activation, expression, and regulation of a series of genes. It is not self-damage under pathological conditions, but rather a form of cell death actively sought by the cell to adapt to its environment. Apoptosis plays a crucial role in multicellular organisms by removing unwanted or abnormal cells, and is significant for organismal evolution, homeostasis, and phylogenetic development. Disorders of the apoptotic process are directly or indirectly related to the occurrence of various diseases such as tumors and autoimmune diseases. Furthermore, apoptosis can be induced by various factors, including natural aging, pathogenic microbial infection, and ionizing radiation. Therefore, the detection of apoptosis is of great importance. The Annexin V assay kit is a commonly used kit for detecting apoptosis. In normal cells, phosphatidylserine is only distributed on the inner side of the cell membrane lipid bilayer. In the early stages of apoptosis, phosphatidylserine is transferred from the inner side of the lipid membrane to the outer side. Annexin V, a phospholipid-binding protein, has a high affinity for phosphatidylserine and binds to the cell membrane of early apoptotic cells via phosphatidylserine residues exposed on the extracellular side. However, the fluorescent molecules of the active dyes in existing annexin V assay kits are prone to photoquenching, and the fluorescent labeling process of annexin V leads to a decrease in its binding affinity to phosphatidylserine. Summary of the Invention

[0003] In view of this, the present invention proposes a cell apoptosis detection kit and its detection method to solve the above problems.

[0004] The technical solution of the present invention is implemented as follows: A cell apoptosis detection kit includes the following raw materials by volume: 2-3 μL annexin V, 0.5-1.5 mL binding solution, 0.5-1.5 mL blocking solution, 2-3 μL reactive dye I, and 4-6 μL reactive dye II. The binding solution is composed of steroidal saponins, calcium ion solution, 4-hydroxyethylpiperazine ethanesulfonic acid, and water in a mass-volume ratio of (0.6-1.0):(0.05-0.1):(0.8-1.2):(96-98) g / mL.

[0005] Furthermore, reactive dye I is fluorescein isothiocyanate, and reactive dye II is propidium iodide or 7-aminoactinomycin D.

[0006] Furthermore, the concentration of reactive dye I is 3 μg / mL-5 μg / mL, and the concentration of reactive dye II is 1 μg / mL-3 μg / mL.

[0007] Furthermore, the calcium ion solution in the binding solution is selected from one of the following: calcium chloride solution, calcium hydroxide solution, calcium gluconate solution, calcium citrate solution, and calcium sulfate solution.

[0008] Furthermore, the pH of the binding solution is 7.0-8.0.

[0009] Furthermore, the blocking solution consists of protamine, sodium chloride solution, and tris(hydroxymethyl)aminomethane hydrochloride solution in a mass-to-volume ratio of (0.6-0.8):(6-8):(6-10) g / mL.

[0010] Furthermore, a detection method for a cell apoptosis detection kit includes the following steps:

[0011] S1. Cell sample collection and washing: Take 1×10⁻⁶ cells... 6 -3×10 6 One cell was placed in a test tube, washed twice with 1.0-1.5 mL of phosphate buffer at 2-8℃, centrifuged, and the supernatant was discarded to obtain the cell sample precipitate.

[0012] S2. Cell preparation: Add 0.5-1.5 mL of binding buffer at 2-8℃ to the cell sample precipitate for resuspending to obtain a cell suspension, and adjust the cell count to 1×10⁻⁶. 6 -3×10 6 Quantity / ml, for later use;

[0013] S3, Annexin V labeling: Mix 2-3 μL of annexin V with 2-3 μL of active dye I, gently vortex to mix, and incubate in the dark to form an annexin V-fluorescein isothiocyanate labeling complex.

[0014] S4. Cell staining and binding: Take 100 μL of cell suspension and add 4-6 μL of annexin V-fluorescein isothiocyanate labeling complex to the cell suspension. Gently vortex to mix and incubate in the dark.

[0015] S5. Blocking and staining: Add 0.5-1.5 mL of blocking solution to the cell suspension after incubation in S4, and add 4-6 μL of active dye II for staining. Gently vortex to mix, incubate in the dark, and obtain a two-color labeled cell sample.

[0016] S6. Flow cytometer parameter settings and detection: Flow cytometer is used to detect two-color labeled cell samples. Set the flow cytometer channels and excitation wavelength, and collect the green and red fluorescence signals of the test samples.

[0017] Furthermore, in S1, the centrifugation speed is 800-1200 r / min, and the centrifugation time is 5-10 min.

[0018] Furthermore, the incubation time in S3, S4, and S5 is 4-6 min, the incubation temperature is 20-25℃, and the vortex rotation speed in S3, S4, and S5 is 600-800 r / min and the vortex rotation time is 5-10 s.

[0019] Furthermore, the flow cytometer in S6 is configured with a green fluorescence channel and a red fluorescence channel. The green fluorescence channel is excited at a wavelength of 488 nm and collects green fluorescence signals at a wavelength of 500-550 nm. The red fluorescence channel is excited at a wavelength of 450-600 nm and collects red fluorescence signals at a wavelength of 570-670 nm.

[0020] Compared with existing technologies, the beneficial effects of this invention are as follows: The steroidal saponins in the binding solution of this invention reduce intermolecular collisions and energy transfer by altering the microenvironment of fluorescent molecules, thereby reducing the interaction between fluorescent molecules and thus reducing fluorescence quenching. The 4-hydroxyethylpiperazine ethanesulfonic acid in the binding solution maintains a stable pH value, helping to reduce acid-base changes in fluorescent molecules and thus reducing fluorescence quenching. The calcium ion solution in the binding solution provides necessary calcium ions, promoting the effective binding of annexin V to phosphatidylserine on the cell membrane. The protamine in the blocking solution of this invention, by binding to non-specific binding sites on the cell surface, reduces the non-specific interaction between these sites and annexin V, helping to ensure the binding of annexin V to phosphatidylserine and improving the binding capacity of annexin V to phosphatidylserine. Sodium chloride, by adjusting the ionic strength of the solution, helps to reduce non-specific binding caused by electrostatic interactions, indirectly improving the binding capacity of annexin V to phosphatidylserine. Tris(hydroxymethyl)aminomethane hydrochloride helps maintain the activity of annexin V and phosphatidylserine by keeping the solution pH stable, thereby enhancing their binding affinity. Detailed Implementation

[0021] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0022] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0023] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0024] Example 1

[0025] A cell apoptosis detection kit comprises the following raw materials in the following volumes: 2 μL annexin V, 0.5 mL binding buffer, 0.5 mL blocking buffer, 2 μL fluorescein isothiocyanate at a concentration of 5 μg / mL, and 4 μL propidium iodide at a concentration of 3 μg / mL. The binding buffer consists of steroidal saponins, calcium ion solution, 4-hydroxyethylpiperazine ethanesulfonic acid, and water in a mass-to-volume ratio of 0.6:0.05:0.8:96 (g / mL), wherein the calcium ion solution is selected from calcium chloride solution, and the pH of the binding buffer is 7.0. The blocking buffer consists of protamine sulfate, sodium chloride solution, and tris(hydroxymethyl)aminomethane hydrochloride solution in a mass-to-volume ratio of (0.6-0.8):(6-8):(6-10) (g / mL).

[0026] The detection method of the above-mentioned apoptosis detection kit includes the following steps:

[0027] S1. Cell sample collection and washing: Take 1×10⁻⁶ cells... 6 One cell was placed in a test tube and washed twice with 1.0 mL of phosphate buffer at 2 °C. The cells were then centrifuged at 800 r / min for 10 min. The supernatant was discarded to obtain the cell sample precipitate.

[0028] S2. Cell preparation: Add 0.5 mL of binding buffer at 2°C to the cell sample precipitate for resuspending to obtain a cell suspension, and adjust the cell count to 1 × 10⁻⁶. 6 Quantity / ml, for later use;

[0029] S3, Annexin V labeling: Mix 2 μL of annexin V with 2 μL of fluorescein isothiocyanate, vortex for 10 s at a vortex speed of 600 r / min, and then incubate at 20 ℃ in the dark for 6 min to form an annexin V-fluorescein isothiocyanate labeled complex.

[0030] S4. Cell staining and binding: Take 100 μL of cell suspension and add 4 μL of annexin V-fluorescein isothiocyanate labeled complex to the cell suspension. Vortex for 10 s at a rotation speed of 600 r / min, and then incubate at 20 ℃ in the dark for 6 min.

[0031] S5. Blocking and staining: Add 0.5 mL of blocking solution to the cell suspension after incubation in S4, and add 4 μL of propidium iodide for staining. Vortex for 10 s at a rotation speed of 600 r / min, and then incubate at 20 ℃ in the dark for 6 min to obtain a two-color labeled cell sample.

[0032] S6. Flow cytometer parameter settings and detection: The flow cytometer was used to detect the two-color labeled cell samples. The flow cytometer channels were set to green fluorescence channel and red fluorescence channel. The excitation wavelength of the green fluorescence channel was 488nm, and the green fluorescence signal with a wavelength of 500-550nm was collected. The excitation wavelength of the red fluorescence channel was 535nm, and the red fluorescence signal with a wavelength of 600-650nm was collected.

[0033] Example 2

[0034] A cell apoptosis detection kit comprises the following raw materials in the following volumes: 2.5 μL annexin V, 1.0 mL binding buffer, 1.0 mL blocking buffer, 2.5 μL fluorescein isothiocyanate at a concentration of 4 μg / mL, and 5 μL 7-aminoactinomycin D at a concentration of 2 μg / mL. The binding buffer consists of steroidal saponins, calcium ion solution, 4-hydroxyethylpiperazine ethanesulfonic acid, and water in a mass-to-volume ratio of 0.8:0.07:1.0:97 g / mL, wherein the calcium ion solution is selected from calcium gluconate solution, and the pH of the binding buffer is 7.5. The blocking buffer consists of protamine sulfate, sodium chloride solution, and tris(hydroxymethyl)aminomethane hydrochloride solution in a mass-to-volume ratio of 0.7:7:8 g / mL.

[0035] The detection method of the above-mentioned apoptosis detection kit includes the following steps:

[0036] S1. Cell sample collection and washing: Take 2×10⁶ cells... 6 One cell was placed in a test tube and washed twice with 1.3 mL of phosphate buffer at 5 °C. The cells were then centrifuged at 1000 r / min for 8 min. The supernatant was discarded to obtain the cell sample precipitate.

[0037] S2. Cell preparation: Add 1.0 mL of binding buffer at 5°C to the cell sample precipitate for resuspending to obtain a cell suspension, and adjust the cell count to 2 × 10⁻⁶. 6 Quantity / ml, for later use;

[0038] S3, Annexin V labeling: Mix 2.5 μL of annexin V with 2.5 μL of fluorescein isothiocyanate, vortex for 7 s at a rotation speed of 700 r / min, and then incubate at 23 °C in the dark for 5 min to form an annexin V-fluorescein isothiocyanate labeled complex.

[0039] S4. Cell staining and binding: Take 100 μL of cell suspension and add 5 μL of annexin V-fluorescein isothiocyanate labeled complex to the cell suspension. Vortex for 7 s at a rotation speed of 700 r / min, and then incubate at 23 ℃ in the dark for 5 min.

[0040] S5. Blocking and staining: Add 1.0 mL of blocking solution and 5 μL of 7-aminoactinomycin D to the cell suspension after incubation in S4. Vortex for 7 seconds at a rotation speed of 700 r / min, and then incubate at 23°C in the dark for 5 minutes to obtain a two-color labeled cell sample.

[0041] S6. Flow cytometry parameter settings and detection: The flow cytometer was used to detect the two-color labeled cell samples. The flow cytometer channels were set to green fluorescence channel and red fluorescence channel. The excitation wavelength of the green fluorescence channel was 488nm, and the green fluorescence signal with a wavelength of 500-550nm was collected. The excitation wavelength of the red fluorescence channel was 546nm, and the red fluorescence signal with a wavelength of 640-670nm was collected.

[0042] Example 3

[0043] A cell apoptosis detection kit comprises the following raw materials in the following volumes: 3 μL annexin V, 1.5 mL binding buffer, 1.5 mL blocking buffer, 3 μL fluorescein isothiocyanate at a concentration of 3 μg / mL, and 6 μL 7-aminoactinomycin D at a concentration of 1 μg / mL. The binding buffer consists of steroidal saponins, calcium ion solution, 4-hydroxyethylpiperazine ethanesulfonic acid, and water in a mass-to-volume ratio of 1.0:0.1:1.2:98 (g / mL), wherein the calcium ion solution is selected from sodium hydroxide calcium solution, and the pH of the binding buffer is 8.0. The blocking buffer consists of protamine sulfate, sodium chloride solution, and tris(hydroxymethyl)aminomethane hydrochloride solution in a mass-to-volume ratio of 0.8:8:10 (g / mL).

[0044] The detection method of the above-mentioned apoptosis detection kit includes the following steps:

[0045] S1. Cell sample collection and washing: Take 3×10⁻⁶ cells... 6 One cell was placed in a test tube and washed twice with 1.5 mL of phosphate buffer at 8 °C. The cells were then centrifuged at 1200 r / min for 5 min. The supernatant was discarded to obtain the cell sample precipitate.

[0046] S2. Cell preparation: Add 1.5 mL of binding buffer at 8°C to the cell sample precipitate for resuspending to obtain a cell suspension, and adjust the cell number to 3 × 10⁻⁶. 6 Quantity / ml, for later use;

[0047] S3, Annexin V labeling: Mix 3 μL of annexin V with 3 μL of fluorescein isothiocyanate, vortex at 800 r / min for 10 s, and then incubate at 25 ℃ in the dark for 4 min to form annexin V-fluorescein isothiocyanate labeled complex.

[0048] S4. Cell staining and binding: Take 100 μL of cell suspension and add 6 μL of annexin V-fluorescein isothiocyanate labeled complex to the cell suspension. Vortex for 10 s at a rotation speed of 800 r / min, and then incubate at 25 ℃ in the dark for 4 min.

[0049] S5. Blocking and staining: Add 1.5 mL of blocking solution to the cell suspension after incubation in S4, and add 6 μL of 7-aminoactinomycin D for staining. Vortex for 10 s at a rotation speed of 800 r / min, and then incubate at 25 ℃ in the dark for 4 min to obtain a two-color labeled cell sample.

[0050] S6. Flow cytometry parameter settings and detection: The flow cytometer was used to detect the two-color labeled cell samples. The flow cytometer channels were set to green fluorescence channel and red fluorescence channel. The excitation wavelength of the green fluorescence channel was 488nm, and the green fluorescence signal with a wavelength of 500-550nm was collected. The excitation wavelength of the red fluorescence channel was 546nm, and the red fluorescence signal with a wavelength of 640-670nm was collected.

[0051] Comparative Example 1

[0052] Compared with Example 2, the difference in this comparative example is that the raw materials of the binding solution do not contain steroidal saponins. The binding solution is composed of calcium ion solution, 4-hydroxyethylpiperazine ethanesulfonic acid, and water with a mass-to-volume ratio of 0.07:1.0:97 g / mL.

[0053] Comparative Example 2

[0054] Compared with Example 2, the difference in this comparative example is that the binding solution is composed of steroidal saponins, calcium ion solution, 4-hydroxyethylpiperazine ethanesulfonic acid, and water in a mass-to-volume ratio of 0.4:0.2:1.5:90 g / mL.

[0055] Comparative Example 3

[0056] Compared with Example 2, the difference in this comparative example is that the kit does not contain blocking solution. It is a cell apoptosis detection kit, which includes the following volumes of raw materials: 2.5 μL annexin V, 1.0 mL binding solution, 2.5 μL fluorescein isothiocyanate at a concentration of 4 μg / mL, and 5 μL 7-aminoactinomycin D at a concentration of 2 μg / mL.

[0057] Comparative Example 4

[0058] Compared with Example 2, this comparative example differs in that the blocking solution does not contain protamine, and the blocking solution is composed of sodium chloride solution and tris(hydroxymethyl)aminomethane hydrochloride solution with a mass-to-volume ratio of 7:8 (g / mL).

[0059] Apoptosis rate detection

[0060] Eight-week-old mice were sacrificed by cervical dislocation, and the thymus was harvested under aseptic conditions. Thymocytes were isolated using the mesh-rubbing method. The thymocytes were centrifuged at 1500 rpm for 10 min in test tubes, washed twice with phosphate-buffered saline (PFS), and the cell concentration was adjusted to 2 × 10⁻⁶ cells / mL. 6 Cells / mL. Azithromycin was added to the above cells to induce apoptosis. The apoptosis rate was detected using transmission electron microscopy. The detection was performed 5 times, and the average result was recorded in Table 1. Then, the above cells were divided into 7 groups, and the apoptosis rate was detected using the cell apoptosis detection kits prepared according to Examples 1-3 and Comparative Examples 1-4, and the detection methods were followed. Each group of samples was tested 5 times, and the average value was recorded in Table 1. Wherein, the apoptosis rate = number of apoptotic cells ÷ total number of cells × 100%, and the cell necrosis rate = number of necrotic cells ÷ total number of cells × 100%.

[0061] Table 1

[0062] Early apoptosis rate Late apoptosis rate Necrosis rate Transmission microscope 26.89 48.89 10.13 Example 1 25.72 47.35 10.04 Example 2 26.88 48.89 10.12 Example 3 25.96 47.59 10.01 Comparative Example 1 20.37 38.71 7.93 Comparative Example 2 21.84 40.62 8.24 Comparative Example 3 21.59 39.85 8.19 Comparative Example 4 23.03 41.59 9.17

[0063] As can be seen from Table 1, the results obtained by the detection kit of the present invention are largely consistent with the results obtained by the transmission microscope. This shows that the detection kit of the present invention can accurately identify early apoptosis, late apoptosis, and necrotic cells. The detection results are accurate and reliable, especially the results of Example 2, which are even closer to the results of the transmission microscope.

[0064] Compared to Comparative Example 1, in Example 2, the steroidal saponins in the binding solution alter the microenvironment of fluorescent molecules, reducing intermolecular collisions and energy transfer, and decreasing interactions between fluorescent molecules. This reduces fluorescence quenching, thus minimizing the weakening of the fluorescence signal caused by quenching. Consequently, flow cytometry struggles to capture sufficient fluorescence signals to accurately identify apoptotic or necrotic cells, ensuring the accuracy of the detection results. Steroidal saponins also alter the local conformation of the cell membrane, promoting the exposure of phosphatidylserine and its binding to annexin V. When the binding capacity of phosphatidylserine to annexin V decreases, the detection of early apoptotic cells becomes inaccurate, leading to a lower detected early apoptosis rate. Therefore, compared to Comparative Example 1, Example 3 shows higher early apoptosis, late apoptosis, and necrosis rates, more closely resembling the results of transmission microscopy, demonstrating higher accuracy.

[0065] Compared with Comparative Example 2, in Example 2, the steroidal saponins, calcium ion solution, 4-hydroxyethylpiperazine ethanesulfonic acid, and water in the binding solution only exert their synergistic effect when they are in a specific ratio. This synergistic effect reduces the fluorescence quenching of reactive dyes and enhances the binding ability of annexin and V phosphatidylserine, thereby improving the detected cell apoptosis rate and cell necrosis rate, and improving the accuracy of the detection results.

[0066] Compared to Comparative Example 3, Example 2, by adding a blocking solution, utilizes protamine, which binds to non-specific binding sites on the cell surface, thereby reducing the non-specific interactions between these sites and annexin V. This helps ensure the binding of annexin V to phosphatidylserine, enhancing the binding capacity of annexin V to phosphatidylserine. Simultaneously, protamine binds to reactive dyes, enhancing the stability of the fluorescence signal and reducing fluorescence quenching. Sodium chloride, by adjusting the ionic strength of the solution, helps reduce non-specific binding caused by electrostatic interactions, indirectly improving the binding capacity of annexin V to phosphatidylserine. Tris(hydroxymethyl)aminomethane hydrochloride, by maintaining the pH stability of the solution, preserves the activity of annexin V and phosphatidylserine, thus contributing to improved binding between them. Therefore, the apoptosis and necrosis rates detected in Example 2 are higher than those in Comparative Example 3, and the results are closer to those obtained using transmission microscopy.

[0067] Compared with Comparative Example 4, Example 2 lacked protamine, and the blocking solution's effect of reducing fluorescence quenching of active dyes and enhancing the binding ability of annexin V to phosphatidylserine was significantly weakened, resulting in a decrease in the detected cell apoptosis rate and cell necrosis rate. This shows that protamine is indispensable in the blocking solution.

[0068] The reagent kit in this invention is 1T, and can be packaged in different sizes such as 10T, 50T, and 100T according to the usage requirements.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An apoptosis detection kit, characterized by comprising: The product comprises the following components by volume: 2-3 μL annexin V, 0.5-1.5 mL binding buffer, 0.5-1.5 mL blocking buffer, 2-3 μL reactive dye I, and 4-6 μL reactive dye II. The binding buffer consists of steroidal saponins, calcium ion solution, 4-hydroxyethylpiperazine ethanesulfonic acid, and water in a mass-to-volume ratio of (0.6-1.0):(0.05-0.1):(0.8-1.2):(96-98) g / mL. Reactive dye I is fluorescein isothiocyanate, and reactive dye II is propidium iodide or 7-aminoactinomycin. D; The concentration of reactive dye I is 3 μg / mL-5 μg / mL, and the concentration of reactive dye II is 1 μg / mL-3 μg / mL; the calcium ion solution in the binding solution is selected from one of calcium chloride solution, sodium hydroxide calcium solution, calcium gluconate solution, calcium citrate solution, and calcium sulfate solution; the pH of the binding solution is 7.0-8.0; the blocking solution is composed of protamine, sodium chloride solution, and tris(hydroxymethyl)aminomethane hydrochloride solution in a mass-to-volume ratio of (0.6-0.8):(6-8):(6-10) g / mL.

2. The detection method of the apoptosis detection kit as described in claim 1, characterized in that, Includes the following steps: S1, cell sample collection and washing: take 1 x 10 6 -3 x 10 6 cells into a test tube, wash twice with 1.0-1.5 mL of phosphate buffer at 2-8°C, centrifugal separation, discard the supernatant, and obtain cell sample precipitate; S2, cell preparation: 0.5-1.5 mL of binding solution at 2-8°C was added to the cell sample precipitate for resuspension, to obtain a cell suspension, and the cell number was adjusted to 1 x 10 6 -3 x 10 6 / ml, standby; S3, Annexin V labeling: Mix 2-3 μL of annexin V with 2-3 μL of active dye I, gently vortex to mix, and incubate in the dark to form an annexin V-fluorescein isothiocyanate labeling complex. S4. Cell staining and binding: Take 100 μL of cell suspension and add 4-6 μL of annexin V-fluorescein isothiocyanate labeling complex to the cell suspension. Gently vortex to mix and incubate in the dark. S5. Blocking and staining: Add 0.5-1.5 mL of blocking solution to the cell suspension after incubation in S4, and add 4-6 μL of active dye II for staining. Gently vortex to mix, incubate in the dark, and obtain a two-color labeled cell sample. S6. Flow cytometer parameter settings and detection: Flow cytometer is used to detect two-color labeled cell samples. Set the flow cytometer channels and excitation wavelength, and collect the green and red fluorescence signals of the test samples.

3. The method of detecting an apoptosis detection kit according to claim 2, wherein In S1, the centrifugal separation speed is 800-1200 r / min, and the centrifugal separation time is 5-10 min.

4. The detection method of the apoptosis detection kit as described in claim 2, characterized in that, The incubation time in S3, S4, and S5 is 4-6 min, the incubation temperature is 20-25℃, the vortex rotation speed in S3, S4, and S5 is 600-800 r / min, and the vortex rotation time is 5-10 s.

5. The detection method of the apoptosis detection kit as described in claim 2, characterized in that, The flow cytometer in S6 is configured with a green fluorescence channel and a red fluorescence channel. The green fluorescence channel is excited at a wavelength of 488 nm and collects green fluorescence signals at a wavelength of 500-550 nm. The red fluorescence channel is excited at a wavelength of 530-550 nm and collects red fluorescence signals at a wavelength of 600-670 nm.