Method for detecting peony pollen cell oxygen consumption rate by using cell energy metabolism analyzer
By optimizing cell adhesion and testing conditions, Seahorse XFe96 method was developed to detect the oxygen consumption rate of peony pollen cells, solving the problem that plant cells cannot be effectively measured, and achieving efficient and economical mitochondrial function and cell metabolism assays.
Patent Information
- Application Number
- CN202510473355.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-16
- Publication Date
- 2025-07-11
AI Technical Summary
The prior art lacks Seahorse cell energy metabolism analyzer detection methods suitable for plant cells, especially because plant cells cannot grow adherently, commercial cell adhesions are expensive and inconvenient to purchase, and lack of plant cell testing systems, making it difficult to effectively determine the mitochondrial function and cell metabolism of pollen cells.
By screening out economical and suitable cell adhesion agents and optimizing cell testing density and drug concentrations, a method for detecting the oxygen consumption rate of peony pollen using Seahorse XFe96 is developed, including using polylysine as cell adhesion agent, configuring appropriate culture medium and drug concentration, and optimizing the testing process.
Effective determination of the oxygen consumption rate of peony pollen cells is achieved. The obtained cellular oxygen consumption rate curve is consistent with the standard curve of animal adherent cells, which reduces the cost of use, provides a reference for mitochondrial function and cell metabolism in plant cells, and is suitable for the determination of different plant cells.
Smart Images
Figure HDA0005360564610000011 
Figure HDA0005360564610000012 
Figure HDA0005360564610000021
Abstract
Description
Technical Field
[0001] The present invention relates to the field of plant cell mitochondrial function detection, and specifically relates to a method for detecting the oxygen consumption rate of peony pollen cells by using a cell energy metabolism analyzer (Seahorse XFe96). Background Art
[0002] Plant mitochondria are complex organelles that participate in many cellular functions such as cell signal transduction, redox regulation, and respiratory metabolism, and are the main sites of cell respiration and bioenergy production. Adenosine triphosphate (ATP) is produced in cells through glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS), and among them, the tricarboxylic acid cycle and OXPHOS only occur in mitochondria. The biological dysfunction of many cells or tissues may stem from the damage of mitochondrial function. At the same time, due to the high adaptability of mitochondria to changes in the cellular environment, the change in its function is also a response to the dysfunction phenotype. Therefore, the determination of mitochondrial function helps to understand the changes and roles of plant mitochondria in a stress environment.
[0003] Generally, the OXPHOS function of mitochondria is evaluated by the oxygen consumption rate (OCR). In the field of plants, common methods for measuring the oxygen consumption rate include the seed respiration analyzer, the oxygen microsensor method, the Clarke electrodes method, and the Seahorse extracellular flux analyzer, etc. Among them, the seed respiration analyzer is suitable for the measurement of large tissues such as seeds, shoot tips, and leaves, etc.; however, as the scale of the measured organism increases, the factors affecting mitochondrial function also increase; the oxygen microsensor method is also applicable to plant tissues, but the measurement throughput is low; the Clarke electrodes method can measure the oxygen content in isolated mitochondria or protoplasts, but the measurement throughput is low, and the isolated mitochondria not only lack the cellular environment but may also be damaged mechanically and osmotically during the isolation process; the Seahorse extracellular flux analyzer is suitable for isolated mitochondria, cells, and tissues, and has a high measurement throughput and can perform quantitative analysis. Measuring mitochondrial function by a non-invasive method at the cellular level has higher physiological relevance. Currently, there is a lack of relevant research on directly measuring the OXPHOS function of mitochondria at the cellular level in the field of plants.
[0004] The Seahorse cell energy metabolism analyzer is a high-throughput metabolic analysis platform with a highly precise, highly sensitive, and highly stable workflow and a full set of mature software functions. Its principle is to apply extracellular flux analysis technology to detect changes in the dissolved oxygen and free proton concentrations within a certain volume around cells over a certain period of time. It has the advantages of not contacting cells, not destroying cell structures, and being able to detect cells in a physiological state in real time. The Seahorse cell energy metabolism analyzer evaluates mitochondrial biological functions by measuring the oxygen consumption rate of cells, including indicators such as basal respiration, proton leak, ATP production, maximal respiration, spare respiration capacity, and non-mitochondrial oxygen consumption. Currently, the Seahorse cell energy metabolism analyzer has been widely used to determine the mitochondrial function and cell metabolism of mammalian cells or tissues, but it is rarely used in plant research. The main reasons restricting its application are that plant cells have cell walls and cannot grow adherently, commercial cell adhesives are expensive and inconvenient to purchase, and there is a lack of plant cell test systems. In addition, the successful determination of the Seahorse cell metabolism analyzer has relatively high requirements for cell density and drug concentration, and it is necessary to repeatedly experiment to explore the optimal conditions. So far, there has been no report on measuring the oxygen consumption rate of pollen cells using the Seahorse cell energy metabolism analyzer. Developing a Seahorse cell energy metabolism analysis method for plant cells is of great significance for understanding the mitochondrial function, cell metabolism status, and bioenergy supply of plant cells.
[0005] Based on the deficiencies and requirements in the above fields, according to the size and characteristics of peony pollen cells, this invention has screened out an economical and suitable cell adhesive, appropriate cell test density, and drug concentration through a large number of experiments, and optimized the test process. The purpose is to provide a method for detecting the oxygen consumption rate of peony pollen cells using a cell energy metabolism analyzer (Seahorse XFe96), so as to provide a suitable, economical, and effective reference guide for the determination of mitochondrial function and cell metabolism of pollen cells. Summary of the Invention
[0006] To achieve the above object, this invention provides a method for detecting the oxygen consumption rate of peony pollen cells using a cell energy metabolism analyzer (Seahorse XFe96).
[0007] First, a method for detecting the oxygen consumption rate of peony pollen cells using a cell energy metabolism analyzer (Seahorse XFe96) according to this invention includes the following steps:
[0008] a. Hydrate the probe plate 12 - 72 h in advance and place it in a 37 °C incubator without CO₂; coat the cell culture microplate with an appropriate concentration of cell adhesion agent solution.
[0009] b. Prepare the basal medium: XF DMEM Medium containing 5 - 25 mM glucose, 0.5 - 4 mM glutamine, and 0.5 - 2 mM sodium pyruvate, and filter - sterilize it after preparation; preferably, filter - sterilize it using a 0.22 - μm filter.
[0010] c. Prepare the pollen suspension: Use PBS buffer to fully suspend the pollen into a uniform pollen suspension.
[0011] d. Replace the PBS medium in the pollen suspension with the basal medium prepared in step b by centrifuging at low speed 2 - 3 times repeatedly.
[0012] e. Fix the pollen cells: Add the pollen suspension with an appropriate density to the cell culture microplate prepared in step a, and let it stand still to make the pollen adhere evenly to the bottom of the wells.
[0013] f. Drug treatment: Add 25 μL of the working solution of the corresponding drug to each of wells A, B, and C in the probe plate, and place the probe plate with the added drugs into the instrument for calibration; among them, the drug in well A: Oligomycin; the drug in well B: FCCP; the drugs in well C: Antimycin and Rotenone.
[0014] g. Place the cell culture microplate with the adhered cells after standing still in step e in a 37 °C incubator without CO₂ and culture for 1 h.
[0015] h. After the probe plate is calibrated, place the cultured cell culture microplate into the machine for on - machine testing.
[0016] i. Set the time program (time event) for each measurement cycle as follows: Mix: 3 min, Wait: 2 min, Measure: 3 min.
[0017] j. After the test is completed, take out the probe plate and the cell culture microplate, and analyze the test results using Wave software.
[0018] Among them, in step a, after coating the cell culture microwell plate with a cell adhesion agent solution at an appropriate concentration for more than 12 h, the cell adhesion agent solution is recovered and the cell culture microwell plate is dried in a laminar flow hood. Preferably, the cell adhesion agent used is poly-D-lysine (PDL), and the preferred concentration of its working solution is 0.1-1 mg / mL, more preferably 0.2 mg / mL, which can effectively play an adhesion role and cause no toxicity to cells.
[0019] Among them, in step c, the pollen cells are fully suspended using 0.01 mol / L PBS solution with a pH of 7.2-7.4. Using 0.01 mol / L PBS (pH 7.4) to fully suspend the pollen cells will not affect the physiological state of the pollen and helps the subsequent measurement to proceed smoothly.
[0020] Among them, in step e, the static adhesion time of the pollen cells to adhere to the wall is 45 min-1 h. Among them, in step f, the concentration of Oligomycin is 0.5 μM, the concentration of FCCP is 1.0 μM, and the concentrations of Antimycin and Rotenone are 10 μM.
[0021] The beneficial effect of the present invention is that for cells that cannot adhere and grow, such as plant cells, a method for detecting the oxygen consumption rate of peony pollen cells using a cell energy metabolism analyzer (Seahorse XFe96) is created. The cell oxygen consumption rate curve obtained by this method is consistent with the Agilent Seahorse XF cell mitochondrial stress test standard curve ( Figure 4 ), and can achieve the effect of adherent animal cells (Yang et al., 2022), and the effect is stable ( Figure 2 ; Figure 3 ; Figure 5 ); the adhesion agent used is easy to purchase, the working solution is easy to prepare and can be recycled, greatly reducing the use cost, and providing a reference for the subsequent determination of mitochondrial function and cell metabolism of different plant cells using the Seahorse cell energy metabolism analyzer. According to the characteristics of peony pollen, the pollen cell density, drug addition concentration and measurement procedure are optimized, providing a method directly for the determination of mitochondrial function and cell metabolism of peony pollen, and also providing a paradigm for the determination of mitochondrial function and cell metabolism of other pollen cells. Description of the Drawings
[0022] Figure 1 Shown are the kinetic rate data (Kinetic Rare Data) of peony pollen cells under different treatments.
[0023] Figure 2 Shown is the change in oxygen consumption rate (OCR) of peony pollen cells under different treatments.
[0024] Figure 3 Shown are the changes in indicators related to mitochondrial biological functions of Paeonia lactiflora pollen cells under different treatments. The mitochondrial biological function indicators include Basal Respiration, Proton Leak, ATP production, Maximal Respiratory, Spare Respiration Capacity, and Non-Mitochondrial Oxygen Consumption.
[0025] Figure 4 Shown is the OCR standard curve of the Agilent Seahorse XF cell mitochondrial stress test (the figure is sourced from www.agilent.com.cn ).
[0026] Figure 5 Shown is the adhesion effect of Paeonia lactiflora pollen under different cell adhesives.
[0027] Figure 6 Shown are the changes in the oxygen consumption rate of Paeonia lactiflora pollen under different cell densities and drug concentrations. (A) 8×10 3 cells / well, 0 μM Oligomycin, 0 μM FCCP, 10 μM Antimycin & Rotenone; (B) 8×10 3 cells / well, 0.5 μM Oligomycin, 0.5 μM FCCP, 10 μM Antimycin & Rotenone; (C) 8×10 3 cells / well, 1 μM Oligomycin, 1 μM FCCP, 10 μM Antimycin & Rotenone (D) 8×10 3 cells / well, 2 μM Oligomycin, 2 μM FCCP, 10 μM Antimycin & Rotenone (E) 1.6×10 4 cells / well, 0 μM Oligomycin, 0 μM FCCP, 10 μM Antimycin & Rotenone (F) 1.6×10 4 cells / well, 0.5 μM Oligomycin, 0.5 μM FCCP, 10 μM Antimycin & Rotenone (G) 1.6×10 4 cells / well, 1 μM Oligomycin, 1 μM FCCP, 10 μM Antimycin & Rotenone (H) 1.6×104 Cells / well, 2 μM Oligomycin, 2 μM FCCP, 10 μM Antimycin & Rotenone (I) 3.2×10 4 Cells / well, 0 μM Oligomycin, 0 μM FCCP, 10 μM Antimycin & Rotenone (J) 3.2×10 4 Cells / well, 0.5 μM Oligomycin, 0.5 μM FCCP, 10 μM Antimycin & Rotenone (K) 3.2×10 4 Cells / well, 1 μM Oligomycin, 1 μM FCCP, 10 μM Antimycin & Rotenone (L) 3.2×10 4 Cells / well, 2 μM Oligomycin, 2 μM FCCP, 10 μM Antimycin & Rotenone (M) 6.4×10 4 Cells / well, 0 μM Oligomycin, 0 μM FCCP, 10 μM Antimycin & Rotenone (N) 6.4×10 4 Cells / well, 0.5 μM Oligomycin, 0.5 μM FCCP, 10 μM Antimycin & Rotenone (O) 6.4×10 4 Cells / well, 1 μM Oligomycin, 1 μM FCCP, 10 μM Antimycin & Rotenone (P) 6.4×10 4 Cells / well, 2 μM Oligomycin, 2 μM FCCP, 10 μM Antimycin & Rotenone. Detailed implementation manners
[0028] The following examples are used to illustrate the present invention, but not to limit the scope of the present invention.
[0029] Using the pollen of Paeonia lactiflora 'Zifeng Chaoyang' collected from the National Flower Engineering Technology Center in Beijing as the research material, different treatments are before cryopreservation (fresh pollen, Fresh) and after cryopreservation (cryopreserved pollen, LN).
[0030] Collect anthers from flower buds at 8:00 - 9:00 in the morning during the natural flowering period. Place the collected anthers on sulfuric acid paper and let them dehisce naturally at room temperature (23 ± 2°C) for 24 h. After the anthers have completely dehisced, pass them through an 80 - mesh sieve and collect the pollen. The collected pollen is randomly divided into two groups, namely the fresh group (Fresh) and the cryopreserved group (LN). The fresh group measures the oxygen consumption rate within one week, and the obtained data is fresh pollen. The cryopreserved group stores the pollen at 0.2 g per cryotube in liquid nitrogen. After 2 months of liquid nitrogen storage, take out the cryotubes, rinse them under running water (15 ± 2°C) for 5 min, and thaw them in water (20 ± 2°C) for 25 min. Measure the oxygen consumption rate of the completely thawed pollen, and the obtained data is cryopreserved pollen.
[0031] Example 1
[0032] A method for detecting the oxygen consumption rate of Paeonia lactiflora pollen cells using a cell energy metabolism analyzer (Seahorse XFe96) includes the following steps:
[0033] 1) Hydrate the probe plate and coat the cell culture microplate in advance
[0034] Hydrate the probe plate with 20 mL of pre - heated XF Calibrant (100840, Agilent, USA) at 37°C at 200 μL per well 12 h in advance and place it in a CO2 - free incubator at 37°C. The hydration time is more than 12 h and within 72 h. After 12 h, repeatedly insert and remove the probe plate to remove air bubbles, and then put it back into the CO2 - free incubator at 37°C for later use; Thaw 10 mg of poly - D - lysine (Poly - D - lysine, PDL, ST508, Beyotime, China) and dissolve it in 2 mL of ultrapure water (ST872, Beyotime, China) to prepare a 5 mg / mL PDL stock solution. Before use, take 400 μL of the 5 mg / mL PDL stock solution and dilute it to 10 mL with ultrapure water to obtain a 0.2 mg / mL PDL working solution. Coat the entire cell culture microplate at 100 μL per well. After overnight coating, recover the PDL working solution and dry the cell culture microplate in a laminar flow hood;
[0035] 2) Prepare the basal medium
[0036] Add 200 μL of 2.5 M glucose (working concentration 10 mM), 500 μL of 200 mM glutamine (working concentration 2 mM), and 500 μL of 100 mM sodium pyruvate (working concentration 1 mM) to 50 mL of XF DMEM Medium (103575, Agilent, USA). After thorough mixing, filter and sterilize it with a 0.22 - μm filter, wrap it with tin foil to avoid light, and pre - heat it at 37°C for later use;
[0037] 3) Prepare the pollen suspension
[0038] Suspend 0.01 g of pollen thoroughly with 1 mL of 0.01 mol / L PBS (pH 7.4) buffer to obtain a uniform pollen suspension with a pollen density of 800 cell / μL;
[0039] 4) Replace the pollen suspension medium
[0040] Use a pipette to aspirate 360 μL of the well-mixed pollen suspension into a 2 mL centrifuge tube, add 1560 μL of the basal medium, shake well and centrifuge at low speed for 5 min. Discard the supernatant. Add 1.5 mL of the basal medium to the precipitated pollen, mix well and centrifuge for 5 min again. Repeat once. Finally, add 1920 μL of the basal medium to the precipitate and mix well. The resulting pollen suspension can be added to 24 wells, with 1.2×10 4 cell per well;
[0041] 5) Fix the pollen cells
[0042] Add 80 μL of the basal medium to the four corners of the cell culture microplate (or the four corners of the selected area) as background wells. Add 80 μL of the pollen suspension from step 4) (note to shake well) to each of the remaining wells. Then slowly add 95 μL of the basal medium to each well (try not to let the liquid flow agitate the pollen) so that the volume of each well system is 175 μL. After adding, let it stand for 1 h to allow the pollen cells to precipitate and adhere to the bottom of the wells;
[0043] 6) Add drugs to the probe plate and calibrate on the instrument
[0044] After thawing and preheating the corresponding drugs at 37°C, add 2.4 μL of 5 mM Oligmycin to 3 mL of the basal medium to obtain a 0.5 μM Oligmycin working solution (well A); add 10.8 μL of FCCP to 3 mL of the basal medium to obtain a 1.0 μM FCCP working solution (well B); add 12 μL of Antimycin and 12 μL of Rotenone to 3 mL of the basal medium to obtain a 10 μM Antimycin and Rotenone working solution (well C); use an auxiliary sample addition plate to add 25 μL of the above drugs to wells A, B, and C of the probe plate respectively. Note that the pipette is perpendicular to the sample addition port and add smoothly without pausing. Place the probe plate with the added drugs into the instrument for calibration;
[0045] 7) Incubate the fixed pollen cells in a 37°C incubator without CO2 for 1 h
[0046] Place the cell culture microplate that has completed static adhesion in step 5) in a 37°C incubator without CO2 and incubate for 1 h;
[0047] 8) Test on the instrument
[0048] When the probe plate calibration in step 6) is completed and the cell culture microplate culture in step 7) is completed, start the test and the facility loading program;
[0049] 9) Set the loading time program
[0050] Set the time program (time event) for each measurement cycle as follows: Mix: 3 min, Wait: 2 min, Measure: 3 min;
[0051] 10) The test is completed
[0052] After the test is completed, take out the probe plate and the cell culture microplate, and analyze the test results using Wave software.
[0053] When performing cell mitochondrial stress tests with a Seahorse cell energy metabolism analyzer, the kinetic rate data ( Figure 1 ) are used to depict the OCR curve, showing the dynamic changes in dissolved oxygen concentration over time ( Figure 2 ); key mitochondrial stress test indicators are calculated based on the OCR curve ( Figure 3 ) to compare the changes in cell mitochondrial biofunction under different treatments. As Figure 2 shown, after cryopreservation of Paeonia lactiflora 'Zifeng Chaoyang' pollen, the overall OCR change trend is significantly lower compared to fresh pollen. Among them, indicators such as basal respiration, proton leak, ATP synthesis, maximal respiration, respiratory potential, and non-mitochondrial oxygen consumption of cryopreserved pollen (LN) are all significantly lower than those of fresh pollen (Fresh), and the decline in respiratory potential is the largest, reaching 86.94% ( Figure 3 ). The results indicate that there is a significant lack of mitochondrial bioenergy supply in 'Zifeng Chaoyang' pollen after cryopreservation. The cell oxygen consumption rate curve obtained by this method is consistent with the Agilent Seahorse XF cell mitochondrial stress test standard curve ( Figure 4 ), and can achieve the effect of adherent animal cells (Yang et al., 2022).
[0054] Example 2
[0055] The pollen used in the experiment was the Paeonia lactiflora 'Zifeng Chaoyang' pollen previously preserved in the cryopreservation library of the research group of Liu Yan from the School of Landscape Architecture, Beijing Forestry University. Except for the parameters that need to be optimized such as the number of pollen cells, the type and concentration of cell adhesives, and the drug concentration, other basic test procedures and conditions are the same as in Example 1.
[0056] 1. Screening experiment on pollen cell adhesives and their concentrations
[0057] At different cell densities of Paeonia pollen (low density: 8×103 Cells / well; high density: 3.2×10 4 Cells / well), use a pipette to add an equal amount of PBS solution (pH 7.4, 2 μL) to each well to simulate the machine drug addition process and observe the movement of pollen grains under a microscope using a 4× objective lens (CX40-RFL, SUNNY GROUP, China) to evaluate the adhesion effect of different cell adhesives. The adhesion results are as Figure 5 shown, 1 mg / mL PDL > 0.2 mg / mL PDL > 0.1 mg / mL PDL > 0.1% gelatin > H2O. According to different plant cell types and sizes, a 0.1 - 1 mg / mL PDL solution can be used to coat the cell culture microplate overnight (coating). After the cell culture microplate is coated, slowly add a uniform pollen cell suspension and let it stand for 45 min - 1 h to effectively adhere plant cells. To avoid the influence of high-concentration adhesives on cells, a 0.2 mg / mL PDL solution was selected as the adhesive for peony pollen cells in this study.
[0058] 2. Comparison of prices of different cell adhesives
[0059] Cell-Tak TM Cell and Tissue Adhesive (Corning, USA) costs $446 for 1 mg (equivalent to approximately 3,252.32 yuan in RMB, calculated at 1 USD = 7.2922 CNY1); the commercial skin adhesive Leukosan (BSN Medical, Australia) costs 163.02 euros for 0.7 mL (equivalent to approximately 1,349.42 yuan in RMB, calculated at 1 EUR = 8.2776 CNY). While polylysine PDL (Beyotime, China) only costs 468 yuan for 10 mg. Among them, Cell-Tak TM and Leukosan are inconvenient to purchase in China and need to contact local distributors for advance ordering, while PDL can be directly shipped from Beyotime's Shanghai factory.
[0060] 3. Screening experiments on pollen cell density and drug concentration parameters
[0061] To optimize the method for detecting the oxygen consumption rate (OCR) of peony pollen using a cell energy metabolism analyzer, the OCR changes of peony pollen at different cell densities and their drug concentrations were tested, and comparative analysis was carried out with reference to the Agilent Seahorse XF cell mitochondrial stress test OCR standard curve. The results are as Figure 6 shown, the appropriate pollen cell density is 8×10 3 ~1.6×10 4Between them, the appropriate concentration of Oligomycin is 0.5 - 1 μM. After adding it, the oxygen consumption of cells shows a downward trend, lower than the basal respiration value, so as to calculate indicators such as basal respiration, ATP production, and proton leakage. The appropriate concentration of FCCP is 0.5 - 1 μM. After adding it, the OCR shows an upward trend, so as to calculate the maximum respiration index; the appropriate concentration of Antimycin & Rotenone is 10 μM, which makes the oxygen consumption show a downward trend, so as to calculate indicators such as respiratory potential and non-mitochondrial oxygen consumption. After comparison, the optimal cell density and drug concentration of peony pollen are selected as: 1.2×10 4 cells / well, Oligomycin 0.5 μM, FCCP 1 μM, Antimycin & Rotenone 10 μM.
[0062] References:
[0063] Yang, J.Q., Fang, L., Lu, H.Y., Liu, C.L., Wang, J., Wu, D., Min, W.H. 2022. Walnut-derived peptide enhances mitophagy via JNK-mediated PINK1 activation to reduce oxidative stress in HT-22 cells. J. Agric. Food Chem. 70: 2630 - 2642.
[0064] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.
Claims
1. A method for detecting the oxygen consumption rate of Paeonia lactiflora pollen cells using a cell energy metabolism analyzer, characterized in that, It includes the following steps: a. Hydrate the probe plate 12 - 72 h in advance and place it in a 37°C incubator without CO₂; coat the cell culture microwell plate with an appropriate concentration of cell adhesion agent solution; b. Prepare the basal medium: XFDMEM Medium containing 5 - 25 mM glucose, 0.5 - 4 mM glutamine, and 0.5 - 2 mM sodium pyruvate, and filter and sterilize it after preparation; c. Prepare the pollen suspension: Use PBS buffer to fully suspend the pollen into a uniform pollen suspension; d. Replace the PBS medium of the pollen suspension with the basal medium prepared in step b by centrifuging at low speed repeatedly 2 - 3 times; e. Fix the pollen cells: Add the pollen suspension with an appropriate density to the cell culture microwell plate prepared in step a, and let it stand to make the pollen adhere evenly to the bottom of the wells; f. Drug treatment: Add 25 μL of the working solution of the corresponding drug to each of wells A, B, and C of the probe plate, and place the probe plate with the added drug into the instrument for calibration; among them, the drug in well A: Oligomycin; the drug in well B: FCCP; the drugs in well C: Antimycin and Rotenone; g. Place the cell culture microwell plate that has completed static adhesion in step e in a 37°C incubator without CO₂ and incubate for 1 h; h. After the probe plate is calibrated, place the cultured cell culture microwell plate into the machine for on - machine testing; i. Set the time program for each measurement cycle to: mixing: 3 min, waiting: 2 min, measuring: 3 min; j. After the test is completed, take out the probe plate and the cell culture microwell plate, and analyze the test results using Wave software.
2. The method for detecting the oxygen consumption rate of peony pollen cells by using a cell energy metabolism analyzer according to claim 1, wherein, After coating the cell culture microwell plate with the cell adhesion agent solution for more than 12 h in step a, recover the cell adhesion agent solution and dry the cell culture microwell plate in a laminar flow hood.
3. A method for detecting the oxygen consumption rate of peony pollen cells using a cell energy metabolism analyzer according to claim 1 or 2, characterized in that, The cell adhesion agent solution in step a is a polylysine solution with a concentration of 0.2 mg / mL (0.1 - 1 mg / mL).
4. A method for detecting the oxygen consumption rate of peony pollen cells using a cell energy metabolism analyzer according to claim 1, characterized in that, The basal medium is XFDMEM Medium containing 10 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate.
5. The method for detecting the oxygen consumption rate of peony pollen cells by using a cell energy metabolism analyzer according to claim 1, characterized in that In step c, use 0.01 mol / L PBS solution with a pH of 7.2 - 7.4 to fully suspend the pollen cells.
6. The method for detecting the oxygen consumption rate of peony pollen cells by using a cell energy metabolism analyzer according to claim 1, characterized in that, In the step e, the density of pollen cells per well is 1.2×10 4 cells.
7. A method for detecting the oxygen consumption rate of peony pollen cells using a cell energy metabolism analyzer according to claim 1, characterized in that, In step e, the static adhesion time of the pollen cells to adhere to the wall is 45 min - 1 h.
8. The method for detecting the oxygen consumption rate of Paeonia lactiflora pollen cells by using a cell energy metabolism analyzer according to claim 1, wherein In step f, the concentration of Oligomycin is 0.5 μM, the concentration of FCCP is 1.0 μM, and the concentrations of Antimycin and Rotenone are 10 μM.