PI flow ploidy analysis kit

Abstract

The invention discloses a PI flow ploidy analysis kit, and relates to the technical field of biological detection. The PI flow ploidy analysis kit provided by the invention integrates a propidium iodide (PI) staining solution and a ribonuclease A (RNase A) reagent. The PI staining solution can be effectively excited by 488nm blue laser widely configured by a flow cytometer, so that the dependence on a special light source is reduced, and the application range of the PI staining solution is greatly widened. The matched RNase A reagent can effectively degrade RNA in cells, eliminates non-specific binding interference of PI and RNA, is helpful for obtaining a clearer DNA content distribution peak graph with a lower background, and improves the accuracy and reliability of ploidy analysis results. According to the invention, necessary PI dye and RNaseA are optimally proportioned in advance and packaged into the ready-to-use reagent, so that the experimental operation is greatly simplified, and the time and labor cost are saved. According to the method, a cell nucleus extracting solution or a staining solution diluent can be further added for ploidy analysis of various cell samples, and the method has the advantages of simplicity in operation and high universality.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a PI flow cytometry ploidy analysis kit. Background Technology

[0002] Flow cytometry, with its high throughput, speed, simplicity, high reproducibility, and multi-parameter analysis capabilities, has become an important technological platform for cell ploidy analysis and cell cycle detection. This technology can process large numbers of cells at once, providing accurate DNA content data in a short time, and has significant application value in fields such as genetic breeding, oncology research, and plant ploidy identification.

[0003] One of the core steps in achieving flow cytometry ploidy analysis is the specific staining of nuclear DNA with a suitable fluorescent dye. An ideal DNA dye should possess high affinity, good spectral characteristics, and its excitation wavelength should be compatible with the widely used laser sources in flow cytometers. Currently, DAPI (4',6-diamidino-2-phenylindole) is commonly used in flow cytometry ploidy analysis due to its stability and specificity in binding to DNA. However, DAPI excitation primarily relies on ultraviolet laser sources (typically 355 nm or 375 nm). This characteristic constitutes a major limitation to its application, as many flow cytometers in use, especially the more common models, are not equipped with ultraviolet lasers, significantly restricting the applicable scenarios for DAPI dyes.

[0004] Therefore, finding a DNA dye that can be effectively excited by more common laser sources (such as the widely deployed 488nm blue laser) is crucial for improving the universality and convenience of flow cytometry ploidy analysis. Propidium iodide (PI), a classic nucleic acid dye, has an excitation spectral peak at approximately 535nm and can be effectively excited by a 488nm laser, emitting at a wavelength of around 617nm (exhibiting red fluorescence). PI works by embedding into base pairs in double-stranded DNA (or RNA) and is not sequence-specific. Notably, PI is a membrane-impermeable dye, meaning it can only enter cells with damaged cell membranes (such as dead or fixed cells) and cannot label live cells. This characteristic also makes it widely used in cell viability detection, apoptosis / necrosis analysis, and microbial viability assessment.

[0005] While PIs theoretically offer good instrument compatibility (excited by a 488nm laser) and relatively low cost, integrating them into ready-to-use kits specifically designed for ploidy analysis remains a challenge in practical applications. Currently, the market offers a very limited selection of commercially available PI kits designed specifically for flow cytometry ploidy analysis, which hinders the convenient application of this technology across a wider range of instrument platforms. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a PI flow cytometry ploidy analysis kit.

[0007] To address the above problems, the present invention proposes the following technical solution:

[0008] In a first aspect, the present invention provides a PI flow cytometry ploidy analysis kit, comprising PI staining solution and RNase A reagent; wherein the concentration of the PI staining solution is 20-80 μg / mL; and the concentration of the RNase A reagent is 20-80 μg / mL.

[0009] Furthermore, the PI staining solution is manufactured by Thermo Fisher Scientific or Sangon Biotech, and the RNase A reagent is manufactured by Takara Bio or Solarbio Technology.

[0010] Furthermore, the concentration of the PI staining solution is 30-60 μg / mL; the concentration of the RNase A reagent is 30-60 μg / mL.

[0011] Based on the staining properties of PI (membrane impermeability) and RNase A treatment, the kit of the present invention can be used for ploidy analysis of various cell samples (such as cultured cells, peripheral blood cells, dissociated tissue cells, plant cell nuclei, etc.) by further adding nuclear extract or staining solution diluent.

[0012] Based on the aforementioned PI flow cytometry ploidy analysis kit, this invention provides a PI flow cytometry ploidy analysis kit for plant cell detection, further comprising a nuclear extract, wherein the nuclear extract comprises the following components:

[0013] Nuclear stabilizer 0.5–8 mmol / L;

[0014] Chelating agent 2–35 mmol / L;

[0015] Na + K + Regulator 80–100 mmol / L;

[0016] Buffer solution 15–250 mmol / L, pH 7.5;

[0017] Triton X-1001 - 2% v / v;

[0018] Sodium metabisulfite 8-12 mmol / L;

[0019] PVP-10 1-2% v / v.

[0020] Furthermore, the nuclear stabilizer is selected from spermine 4HCl or MgCl2·6H2O; the chelating agent is selected from Na2EDTA or sodium citrate; and the buffer solution is selected from Tris-HCl or MOPS.

[0021] Furthermore, the cell nuclear extract is composed of the following components;

[0022] MgCl2·6H2O 2.0–6.0mmol / L;

[0023] Na2EDTA 1.0–3.0 mmol / L;

[0024] Na + K + Regulator 75–110 mmol / L;

[0025] MOPS buffer 15–25 mmol / L, pH 7.5;

[0026] Triton X-100 1-2% v / v;

[0027] Sodium metabisulfite 8-12 mmol / L;

[0028] PVP-10 1-2% v / v.

[0029] Based on the aforementioned PI flow cytometry ploidy analysis kit, the present invention provides a PI flow cytometry ploidy analysis kit for animal cell detection, which also includes a staining solution diluent.

[0030] It should be noted that the staining solution dilution buffer helps maintain the basic environment for PI staining, ensures PI-DNA binding efficiency and RNase A activity, and avoids interference from residual RNA; furthermore, it helps maintain the stability of the cell nucleus morphology after fixation, prevents DNA peak broadening caused by osmotic rupture, and improves the accuracy of the analysis results.

[0031] Furthermore, the staining solution diluent contains 1×PBS and 0-0.05% v / v Triton X-100.

[0032] Furthermore, the staining solution is diluted with 1×PBS.

[0033] Another aspect of the present invention provides the application of a PI flow cytometry ploidy analysis kit containing PI staining solution, RNase A reagent and cell nuclear extract in the detection and analysis of plant cell ploidy.

[0034] Another aspect of the present invention provides the application of a PI flow cytometry ploidy analysis kit containing PI staining solution, RNase A reagent and staining solution diluent in the detection and analysis of ploidy in animal cells.

[0035] Compared with the prior art, the technical effects achieved by the present invention include:

[0036] The PI flow cytometry ploidy analysis kit provided by this invention integrates propidium iodide (PI) staining solution and ribonuclease A (RNase A) reagent. The PI staining solution can be effectively excited by a 488nm blue laser, widely available in flow cytometers, reducing dependence on special light sources (such as ultraviolet lasers) and greatly expanding its application range, making it easier for more laboratories to perform ploidy analysis. The accompanying RNase A reagent effectively degrades intracellular RNA, eliminating interference from non-specific binding of PI to RNA and ensuring that the fluorescence signal mainly originates from the DNA-PI complex. This helps to obtain clearer DNA content distribution peaks with lower background, improving the accuracy and reliability of ploidy analysis results.

[0037] Furthermore, this invention pre-optimizes the ratio of the necessary PI dye and RNase A and packages them as ready-to-use reagents, eliminating the tedious steps of users having to prepare, experiment with concentrations and combinations themselves. Users only need to perform sample processing according to standard procedures (such as cell fixation / permeabilization), add the reagents provided in this kit for staining and digestion reactions, and the sample preparation is completed, greatly simplifying experimental operations and saving time and labor costs.

[0038] Based on the staining characteristics of PI (membrane impermeability) and RNase A treatment, the kit of the present invention can be used for ploidy analysis of various cell samples (such as cultured cells, peripheral blood cells, dissociated tissue cells, plant cell nuclei, etc.) by further adding cell nuclear extract or staining solution dilution, and has the advantages of simple operation and strong versatility.

[0039] The PI flow cytometry ploidy analysis kit provided by this invention has a lower cost of PI dye. The PI dye and RNase A are pre-optimized in ratio and packaged as ready-to-use reagents, which reduces the risk of waste caused by improper reagent dispensing, storage or self-preparation errors. It provides a more convenient, reliable and widely applicable ploidy analysis tool for scientific research and clinical testing. Attached Figure Description

[0040] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 The results show the accelerated stability of tomato leaf samples on day 12.

[0042] Figure 2 The results are from the accelerated stability test of the chicken blood sample on day 15. Detailed Implementation

[0043] The invention is further illustrated below by specific application examples, but these examples do not limit the invention to the scope of the specific applications described. Experimental methods not specifying particular conditions in the following specific application examples should be performed according to conventional methods and conditions, or as selected in the product instructions. Unless otherwise specified, temperature generally refers to reactions conducted at room temperature; in this invention, room temperature refers to 16°C to 30°C.

[0044] Unless otherwise specified, the experimental methods used in the following application examples are conventional methods.

[0045] Unless otherwise specified, all materials and reagents used in the following application examples are commercially available.

[0046] Example 1

[0047] This embodiment provides a PI flow cytometry ploidy analysis kit for plant cell ploidy detection and analysis, hereinafter referred to as: PI flow cytometry ploidy analysis kit (plant). The kit includes nuclear extract, PI staining solution, and RNase A reagent; the concentration of the PI staining solution is 20-80 μg / mL; the concentration of the RNase A reagent is 20-80 μg / mL.

[0048] The flow cytometry detection methods for plant samples are described below:

[0049] 1. Cut fresh leaves and place them in a culture dish. Add 1 mL of nuclear extract and use a blade to quickly and vertically cut the tissue for about 2 minutes (this operation should always be performed on ice) until there are no visible leaf pieces. Then place the culture dish in a 4°C refrigerator and incubate for 5 minutes.

[0050] 2. Take the lysis buffer, filter it through a 50μm / 35μm nylon mesh into a flow cytometer, and centrifuge at 1000r / min for 5min;

[0051] 3. Gently remove the centrifuge tube and slowly aspirate the supernatant (leaving approximately 100μL to 120μL). Add 300μL of reagents (nuclear extraction solution: 285μL, PI staining solution: 15μL, RNase A: 1.5μL).

[0052] 4. Stain in the dark for 25 minutes, then analyze using flow cytometry.

[0053] Note: The required sample volume, weight, incubation time, and other conditions may vary depending on the plant species or different plant parts.

[0054] In some embodiments, the nuclear extract consists of the following components:

[0055] Nuclear stabilizer 0.5–8 mmol / L;

[0056] Chelating agent 2–35 mmol / L;

[0057] Na + K + Regulator 80–100 mmol / L;

[0058] Buffer solution 15–250 mmol / L, pH 7.5;

[0059] Triton X-100 1-2% v / v;

[0060] Sodium metabisulfite 8-12 mmol / L;

[0061] PVP-10 1-2% v / v.

[0062] In some embodiments, the nuclear stabilizer is selected from spermine 4HCl or MgCl2·6H2O; the chelating agent is selected from Na2EDTA or sodium citrate; and the buffer is selected from Tris-HCl or MOPS.

[0063] In some embodiments, the cell nuclear extract formulation is as follows:

[0064] MgCl2·6H2O 2.0–6.0mmol / L;

[0065] Na2EDTA 1.0–3.0 mmol / L;

[0066] Na + K + Regulator 75–110 mmol / L;

[0067] MOPS buffer 15–25 mmol / L, pH 7.5;

[0068] Triton X-100 1-2% v / v;

[0069] Sodium metabisulfite 8-12 mmol / L;

[0070] PVP-10 1-2% v / v.

[0071] In this embodiment of the invention, the formulation of the cell nucleus extract is as follows:

[0072] MgCl2·6H2O 4.0mmol / L;

[0073] Na2EDTA 2.0mmol / L;

[0074] NaCl 86mmol / L;

[0075] MOPS buffer 20.0 mmol / L, pH 7.5;

[0076] Triton X-100 1% v / v;

[0077] Sodium metabisulfite 10 mmol / L;

[0078] PVP-10 1% v / v.

[0079] In this embodiment of the invention, the PI staining solution is produced by Thermo Fisher Scientific or a manufacturer, and the RNase A reagent is produced by Takara or Solarbio.

[0080] To verify the effect of different brands of reagents (PI staining solution + RNase A) on the results of plant ploidy analysis, the final concentration of PI staining solution and RNase A was 50 μg / mL. In this example, a crossover experiment was designed, with three replicates per group (tomato leaf samples). The optimal combination was screened by evaluating the stability of key indicators (CV% and RCS). The results are shown in Table 1.

[0081] Table 1. Detection results of different brands of reagents (PI staining solution + RNase A) combinations.

[0082]

[0083] The results showed that both Takara and Solarbio's RNase A had good CV values, but Solarbio's RNase A required repeated freeze-thaw cycles, which may affect the ease of operation. Sangon Biotech's PI staining solution did not show a significant difference in CV values ​​compared to Thermo Fisher Scientific's PI, and considering its cost advantage, it can be the preferred choice.

[0084] Taking all factors into consideration, this embodiment of the invention uses a combination of Sangon Biotech PI staining solution and Takara RNase A to further explore its applicable concentration.

[0085] PI staining solution:

[0086] The bio-PI powder was prepared into a 1 mg / mL stock solution, which was then diluted to final solutions of 25 μg / mL, 50 μg / mL, and 75 μg / mL. The final concentration of RNase A was 50 μg / mL. The samples were tomato leaves, and the results are shown in Table 2 below.

[0087] Table 2. Detection results of PI staining solutions at different concentrations

[0088]

[0089] The results showed that there was no significant difference in CV values ​​among different concentrations of PI staining solution. In this embodiment of the invention, a concentration of 50 μg / mL was selected.

[0090] RNase A:

[0091] Takara RNase A was prepared into final solutions of 25 μg / mL, 50 μg / mL, and 75 μg / mL, with a final PI concentration of 50 μg / mL. The samples were tomato leaves, and the results are shown in Table 3 below.

[0092] Table 3. Detection results of RNase A at different concentrations

[0093]

[0094] The results showed that, except for 0 μg / mL, there was no significant difference in CV values ​​among the groups. In this example, a concentration of 50 μg / mL was selected.

[0095] In the following tests, the PI flow cytometry ploidy analysis kit (plant) contains nuclear extract, 50 μg / mL bioengineered PI staining solution, and 50 μg / mL Takara RNase A.

[0096] (1) Universality test

[0097] The PI flow cytometry ploidy analysis kit (plant) of this invention was used as the experimental group in triplicate. Different plant samples were tested, and the mean values ​​of the three replicates for the experimental group were calculated. The results are shown in Table 4.

[0098] Table 4. Universality test results of the PI flow cytometry ploidy analysis kit (plant).

[0099]

[0100]

[0101] The results showed that the CV values ​​of the kit of the present invention were reliably repeatable, the test results were accurate, and it had good universality.

[0102] (2) Accelerated stability test

[0103] Three different batches of the PI flow cytometry ploidy analysis kit (plant) of the present invention were stored in an incubator at 37°C. Samples were taken and tested at the times specified in Table 5. The test sample was tomato leaves, and the test results are shown in Table 5.

[0104] Table 5. Stability test results of the PI flow cytometry ploidy analysis kit (plant).

[0105]

[0106] Figure 1 The results of accelerated stability testing of tomato leaves on day 12 are presented. The results show that even after 15 days of accelerated testing at 37°C, the CV value remained less than 8%, indicating that the PI flow cytometry ploidy analysis kit (plant) of this invention has good stability.

[0107] Example 2

[0108] This embodiment provides a PI flow cytometry ploidy analysis kit, which is used for ploidy detection and analysis of animal cells, hereinafter referred to as: PI flow cytometry ploidy analysis kit (animal). The kit includes staining solution diluent, PI staining solution, and RNase A.

[0109] The flow cytometry detection methods for different animal tissue samples are described below:

[0110] I. Animal tissue samples

[0111] 1. After placing the tissue into a petri dish, add 1 mL of PBS, grind it into a homogenate, filter it into a flow cytometer through a 50 μm / 35 μm nylon mesh, and centrifuge at 1000 r / min for 15 min.

[0112] 2. Add 1 mL of PBS, centrifuge at 1000 rpm for 15 min, discard the supernatant, and repeat once more;

[0113] 3. Add 1 mL of 70% v / v ethanol pre-cooled in an ice bath, gently mix by pipetting, fix at 4°C for at least 2 h, centrifuge at 1000 r / min for 15 min, and discard the supernatant.

[0114] 4. Add 1 mL PBS, centrifuge at 1000 r / min for 15 min, discard the supernatant, and gently tap the centrifuge tube to disperse the cells;

[0115] 5. Add 300 μL of reagent (staining solution dilution: 285 μL, PI staining solution: 15 μL, RNase A: 1.5 μL), incubate at room temperature for 30 min, and then analyze using flow cytometry.

[0116] II. Blood Samples

[0117] 1. Take 100 μL of anticoagulated blood, add 1 mL of PBS, mix gently, centrifuge at 300 × g for 5 minutes, discard the supernatant, and repeat once;

[0118] 2. Add 1 mL of 70% ethanol pre-cooled in an ice bath, and fix at 4°C overnight (or at least 2 hours);

[0119] 3. Centrifuge to remove ethanol (300×g, 5 minutes), and wash twice with PBS.

[0120] 4. Add 300 μL of DNA reagent (staining solution dilution: 285 μL, PI staining solution: 15 μL, RNase A: 1.5 μL), incubate at room temperature for 30 min, and then perform the detection.

[0121] In this embodiment of the invention, the PI staining solution is produced by Thermo Fisher Scientific or a manufacturer, and the RNase A reagent is produced by Takara or Solarbio.

[0122] To verify the effect of different brands of reagents (PI staining solution + RNase A) on the results of animal ploidy analysis, the final concentration of PI staining solution and RNase A was 50 μg / mL, and the staining solution was diluted with 1×PBS. In this example, a crossover experiment was designed, with three replicates (chicken blood) for each group. The optimal combination was screened by evaluating the stability of key indicators (CV% and RCS). The results are shown in Table 6.

[0123] Table 6. Detection results of different brands of reagents (PI staining solution + RNase A) combinations.

[0124]

[0125] The results showed that all the above combinations exhibited excellent fit, but the repeated freeze-thaw cycles required for Solarbio RNase A may affect the ease of operation; Sangon Biotech PI staining solution and Thermo Fisher Scientific PI showed no significant difference in CV value, and considering its cost advantage, it can be the preferred choice.

[0126] Taking all factors into consideration, this embodiment of the invention uses a combination of Sangon Biotech PI staining solution and Takara RNase A to further explore its applicable concentration.

[0127] PI staining solution:

[0128] The bio-PI powder was prepared into a 1 mg / mL stock solution, which was then diluted to final solutions of 25 μg / mL, 50 μg / mL, and 75 μg / mL. The final concentration of RNase A was 50 μg / mL. The staining solution was diluted with 1×PBS. Chicken blood was used as the sample for testing. The results are shown in Table 7 below.

[0129] Table 7. Detection results of PI staining solutions at different concentrations

[0130]

[0131] The results showed that there was no significant difference in CV values ​​among different concentrations of PI staining solution. In this embodiment of the invention, a concentration of 50 μg / mL was selected.

[0132] RNase A:

[0133] Takara RNase A was prepared into final solutions of 25 μg / mL, 50 μg / mL, and 75 μg / mL. The final concentration of PI was 50 μg / mL. The staining solution was diluted with 1×PBS. Chicken blood was used as the sample for testing. The results are shown in Table 8 below.

[0134] Table 8. Detection results of RNase A at different concentrations

[0135]

[0136] The results showed that, except for 0 μg / mL, there was no significant difference in CV values ​​among the groups. In this example, a concentration of 50 μg / mL was selected.

[0137] The staining solution diluent in this embodiment contains 1×PBS and 0-0.05% Triton X-100. The effect of the specific formulation and concentration of the staining solution diluent on the detection results was investigated below. Experiments were conducted using 1×PBS with 0%, 0.01%, and 0.03% Triton X-100 added, respectively. The sample was chicken blood, the PI staining solution was 50 μg / mL, and the RNase A was 50 μg / mL of Takara RNase A. The results are shown in Table 9.

[0138] Table 9. Detection results of different staining solution dilutions

[0139]

[0140] The results showed that the amount of Triton X-100 added had no significant effect on the detection results. In this embodiment of the invention, 1×PBS was used as the staining solution dilution.

[0141] In the following tests, the PI flow cytometry ploidy analysis kit (animal) contains 1×PBS staining solution dilution, 50 μg / mL Sangon PI staining solution, and 50 μg / mL Takara RNase A.

[0142] (1) Universality test

[0143] The PI flow cytometry ploidy analysis kit (animal) of this invention was used as the experimental group in triplicate. Different animal samples were tested, and the mean values ​​of the three replicates for the experimental group were calculated. The results are shown in Table 10.

[0144] Table 10. Universality test results of the PI flow cytometry ploidy analysis kit (animals)

[0145]

[0146] The results showed that in representative aquatic animals such as clams, Litopenaeus vannamei, and carp, the CV value of the kit of this invention was stable at 3.91%–6.58%, with good cell cycle peak separation, accurate test results, and good universality.

[0147] (2) Stability test

[0148] Three different batches of the PI flow cytometry ploidy analysis kit (animal) of the present invention were stored in an incubator at 37°C. Samples were taken and tested at the times specified in Table 11. The test sample was chicken blood. The test results are shown in Table 11.

[0149] Table 11 Stability test results of the PI flow cytometry ploidy analysis kit (animal).

[0150]

[0151]

[0152] Figure 2 The results of accelerated stability testing of chicken blood samples on day 15 are presented. The results show that even after 15 days of accelerated testing at 37°C, the CV value remained less than 8%, indicating that the PI flow cytometry ploidy analysis kit (animal) of this invention has good stability.

[0153] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0154] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A PI flow cytometry ploidy analysis kit, characterized in that, It includes PI staining solution and RNase A reagent; the concentration of PI staining solution is 20-80 μg / mL; the concentration of RNase A reagent is 20-80 μg / mL.

2. The PI flow cytometry ploidy analysis kit as described in claim 1, characterized in that, The PI staining solution was manufactured by Thermo Fisher Scientific or a manufacturer of RNase A reagent, and the RNase A reagent was manufactured by Takara or Solarbio.

3. The PI flow cytometry ploidy analysis kit as described in claim 1, characterized in that, The concentration of the PI staining solution was 30-60 μg / mL; the concentration of the RNase A reagent was 30-60 μg / mL.

4. The PI flow cytometry ploidy analysis kit according to any one of claims 1-3, characterized in that, It also includes a nuclear extract, which is composed of the following components: Nuclear stabilizer 0.5–8 mmol / L; Chelating agent 2–35 mmol / L; Na + K + Regulator 80–100 mmol / L; Buffer solution 15–250 mmol / L, pH 7.5; Triton X-1001 - 2% v / v; Sodium metabisulfite 8-12 mmol / L; PVP-101-2% v / v.

5. The PI flow cytometry ploidy analysis kit as described in claim 4, characterized in that, The nuclear stabilizer is selected from spermine 4HCl or MgCl2·6H2O; the chelating agent is selected from Na2EDTA or sodium citrate; and the buffer is selected from Tris-HCl or MOPS.

6. The PI flow cytometry ploidy analysis kit as described in claim 5, characterized in that, The cell nucleus extract is composed of the following components; MgCl2·6H2O 2.0–6.0mmol / L; Na2EDTA 1.0–3.0 mmol / L; Na + K + Regulator 75–110 mmol / L; MOPS buffer 15–25 mmol / L, pH 7.5; Triton X-1001 - 2% v / v; Sodium metabisulfite 8-12 mmol / L; PVP-101-2% v / v.

7. The PI flow cytometry ploidy analysis kit according to any one of claims 1-3, characterized in that, It also includes staining solution diluent.

8. The PI flow cytometry ploidy analysis kit as described in claim 7, characterized in that, The staining solution diluent contains 1×PBS and 0-0.05% Triton X-100.

9. The application of the PI flow cytometry ploidy analysis kit as described in any one of claims 1-3 or the PI flow cytometry ploidy analysis kit as described in any one of claims 4-6 in the detection and analysis of plant cell ploidy.

10. The application of the PI flow cytometry ploidy analysis kit as described in any one of claims 1-3 or the PI flow cytometry ploidy analysis kit as described in any one of claims 7-8 in the detection and analysis of ploidy in animal cells.

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