Method for measuring energy metabolism of chlorella mitochondrial stress cells

Abstract

The invention belongs to the technical field of algae development experiments, and particularly relates to a method for measuring energy metabolism of chlorella mitochondrial stress cells. According to the method, an Agilent cell energy meter XF24 is utilized, the setting of an Agilent default experimental program is optimized, and a mixing part in all reading programs is canceled; the drug concentrations of the three targeting drugs added into the mitochondrial electron transport chain are designed through a large number of research tests, and the number of measurement cycles (5-6 times) after the drugs are added is increased, so that the oxygen consumption rate of cells is measured to obtain key parameters reflecting the mitochondrial function, and the measurement result is more accurate. According to the method, a chlorella mitochondrial pressure curve graph is given, and a new research direction is provided for energy metabolism of algae cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of algal development experiments, and particularly relates to a method for measuring Chlorella pyrenoidosa mitochondrial stress cell energy metabolism. BACKGROUND

[0002] Oxidative phosphorylation, as a cell energy metabolism pathway, is mainly carried out in the inner membrane of mitochondria, is the process of providing a large amount of ATP synthesized by ADP and inorganic phosphate through the respiratory chain by the energy released when the material is oxidized in the body. The process is the main energy source for the life activities of aerobic cells and is the main way for biological ATP production. 80% of the energy required for cell life activities comes from mitochondria, and therefore mitochondria are also called the power plant of cells.

[0003] The Agilent Seahorse XF analyzer can monitor living cells in a multi-well plate in real time and dynamically, and simultaneously measure the oxygen consumption rate (OCR) of the cells in the well plate to study the mitochondrial respiration of the cells. Compared with the previous experimental method for measuring mitochondria, the analyzer can measure the energy of mitochondria without damaging the mitochondria, can comprehensively investigate the function of mitochondria by adding different drugs, can reflect the real physiological state of mitochondria in cells, and can observe the dynamic changes of the function of mitochondria in real time. At the same time, the changes in the metabolic state of cells under different stimulation conditions can be observed, and the changes and conversion of biological energy of cells can be more clearly displayed.

[0004] The method is to measure the oxygen consumption rate of cells by sequentially adding target drugs of the mitochondrial electron transport chain (ETC) to obtain key parameters reflecting the function of mitochondria. After measuring the basal respiration of cells, the first drug added is oligomycin, which inhibits ATP synthase (i.e., complex V), causing mitochondrial respiration or OCR to decrease. This part of the decreased OCR is the ATP-related respiration.

[0005] The second drug added is carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), which can destroy the proton gradient and mitochondrial membrane potential, causing electrons to be transmitted in the ETC without restriction, and the oxygen consumption of complex IV reaches the maximum, i.e., the maximal respiration. The difference between the maximal respiration and the basal respiration is the spare respiratory capacity of the cells, which represents the ability of the cells to respond to an increase in energy demand or under stress.

[0006] The last drug added is a rotenone / antimycin A mixture (Rot / AA). Rotenone is an inhibitor of complex I and antimycin A is an inhibitor of complex III. These two drugs shut down mitochondrial respiration, allowing the calculation of non-mitochondrial oxygen consumption driven by activities outside the mitochondria. The residual oxygen consumption after subtracting the basal respiration from the ATP-related respiration is the proton leak, which is the part of oxygen consumption that is not coupled to ATP synthesis. The proton leak can be used as a marker of mitochondrial damage and can also be considered as a mechanism to regulate mitochondrial ATP synthesis.

[0007] The instrument requires that various cell types and tissue samples are inoculated or fixed on the bottom of the wells of a 24-well XF microplate or an insulin capture plate. The probe plate is provided with a drug injection port. After the instrument reads the basal OCR value for three times according to the default program recommended by Agilent, the system injects three kinds of drugs into the analysis wells according to the set injection order. After each injection, the data is generally read for three times. The program for reading all data is as follows: mixed program for 3 min; wait for 2 min for the cell state to be stable; the instrument lowers the sensor to 200 μm above the bottom of the well, and reads the change of oxygen and pH data in about 7 μL of the transient microchamber (28 μL for the islet plate) for 3 min.

[0008] However, the conventional Seahorse experiment in the prior art is generally used for animal cells or adherent cells, but has not been applied to the research field of Chlorella. Compared with animal cells, Chlorella is a microalgae and belongs to a photosynthetic autotroph. Unlike conventional mammalian cells, Chlorella contains a cell wall and grows in suspension in the culture medium, and has poor adhesion effect. During the process of reading data by the instrument, the Chlorella at the bottom of the plate is resuspended in the liquid, and the oxygen result in the transient microchamber measured by the instrument is inaccurate, and the change of the mitochondrial function parameter of Chlorella cannot be characterized.

[0009] Therefore, researchers generally use JC-1 staining method and ROS content measurement to characterize the change of algal mitochondrial function, but the JC-1 staining method can only characterize the cell growth state such as apoptosis through the change of mitochondrial membrane potential, and cannot reflect the change process of oxidative phosphorylation in the mitochondria. The ROS content in the plant body is mainly produced in organelles such as mitochondria, chloroplasts and peroxisomes, so the detection of ROS content cannot accurately characterize the change of mitochondrial function. SUMMARY

[0010] The present application aims at providing a method for measuring the cell energy metabolism of Chlorella mitochondria stress by using Agilent Cell Energy Meter XF24 in view of the above-mentioned defects.

[0011] Although the Agilent Cell Energy Meter XF24 has been maturely applied to the cell mitochondria respiration research experiment of animal cells or adherent cells, the present application finds that, when the Agilent Cell Energy Meter XF24 is used for the algae cell energy metabolism research, especially the measurement of the cell energy metabolism of Chlorella mitochondria stress, the first technical problem is that Chlorella is easy to suspend in the measurement process, and the adherent effect is poor; then, due to the fact that Chlorella contains a cell wall, the reaction time of the drug with Chlorella is slow, and the measurement efficiency is extremely low; more seriously, the experimental results are extremely unstable, such as unstable signal, no drug response and poor repeatability, and ideal experimental results of the metabolic energy of algae cells cannot be obtained.

[0012] Therefore, the present application makes a large number of explorations and innovations for the test of Chlorella cell energy, comprehensively considers the cell density of Chlorella, verifies the concentration and treatment time of the drug and the compatibility of the culture medium, and obtains the method for measuring the cell energy metabolism of Chlorella mitochondria stress.

[0013] The specific scheme is as follows:

[0014] A method for measuring the cell energy metabolism of Chlorella mitochondria stress, comprising the following steps:

[0015] (1) Preparation: 24h before the measurement experiment, start the Agilent Cell Energy Meter XF24 software Wave, and warm up to 25-28℃;

[0016] and add 1mL of XF Calibrant calibration solution with pH of 7.4 in each well of the hydration plate, and place the whole probe plate device in a CO2-free cell incubator at 25-28℃, and hydrate for 12h.

[0017] (2) Add 50-55μL of Cell-Tak coating solution with a concentration of 22-22.5μg / mL in each well of the cell culture plate;

[0018] After incubation at room temperature for 20-25min, wash each well with sterile water for 2 times, and wait for use.

[0019] (3) Count the Chlorella by using a flow cytometer, and adjust the volume of Chlorella so that the total number of Chlorella cells in 1mL of Chlorella liquid is 1.5×10 7 -2.5×10 7The Chlorella cell density range is designed to meet the basic requirements of ensuring that the basic OCR value of the cells is 200-300 pmol / min, and the cell number at the bottom of the well plate is evenly distributed without accumulation, and the coverage rate at the bottom of the plate is 80%-90%.

[0020] (4) Take 100 μL of Chlorella liquid containing a total of 1.5 x 10 6 -2.5 x 10 6 cells and centrifuge the cells at 500 x g for 5 min at room temperature. Then, the cell culture plate is incubated at room temperature for 25-30 min to ensure that the Chlorella is completely attached to the bottom. The 500 x g refers to the centrifugal force, i.e., the centrifugal force is 500 times the gravity.

[0021] (5) Use XF RPMI medium to configure the drug working solution of oligomycin, carbonyl cyan-4(trifluoromethoxy) phenylhydrazone, and rotenone / antimycin A mixture. RPMI medium is a type of cell culture medium developed by Roswell Park Memorial Institute.

[0022] (6) Take the hydrated probe plate from the 25-28°C CO2-free cell incubator, and add oligomycin to the Port A well of the probe plate; add carbonyl cyan-4(trifluoromethoxy) phenylhydrazone to the Port B well of the probe plate; add rotenone / antimycin A mixture to the Port C well of the probe plate; after the instrument injects the drugs according to the program settings, the final concentration of oligomycin in each well of the cell culture plate is 25-35 μM, the final concentration of carbonyl cyan-4(trifluoromethoxy) phenylhydrazone is 55-65 μM, and the final concentration of rotenone / antimycin A mixture is 25-35 μM.

[0023] (7) Set the cell energy metabolism instrument program, open the mitochondrial stress experiment template in the software, and first set the basic respiration OCR measurement for 3 cycles;

[0024] Then, the instrument injects the oligomycin drug working solution and measures 6 cycles;

[0025] The instrument injects the carbonyl cyan-4(trifluoromethoxy) phenylhydrazone drug working solution and measures 5 cycles;

[0026] The instrument injects the rotenone / antimycin A mixture drug working solution and measures 4-5 cycles;

[0027] Each cycle program is 2 min waiting and 3 min measurement.

[0028] (8) After the program is set, place the drug-added probe plate and the hydrated plate into the instrument for probe calibration.

[0029] (9) In the process of waiting for calibration, add RPMI medium to each well of the cell culture plate, and check the adhesion of chlorella under a microscope after standing for 1 min. After adhesion is completed, react in the dark at room temperature for 20 min.

[0030] (10) After calibration is completed, replace the hydration plate with the cell culture plate treated in the dark, and perform detection.

[0031] (11) The Agilent cell energy meter XF24 software Wave automatically calculates the chlorella mitochondrial stress curve.

[0032] In the present application, the method for determining the cell energy metabolism of chlorella mitochondrial stress, in step (1), the temperature is raised to 28℃.

[0033] In the present application, the method for determining the cell energy metabolism of chlorella mitochondrial stress, in step (2), the concentration of Cell-Tak coating solution is 22.4 μg / mL, and the amount used is 50 μL; the amount of sterile water used is 200 μL.

[0034] In the present application, the method for determining the cell energy metabolism of chlorella mitochondrial stress, in step (3), the total number of chlorella cells per 1 mL is 2×10 7 In step (4), the total number of chlorella cells in 100 μL of chlorella liquid is 2×10 6

[0035] In the present application, the method for determining the cell energy metabolism of chlorella mitochondrial stress, in step (5), the working solution concentration of oligomycin is 298 μM; the working solution concentration of carbonyl cyan-4 (trifluoromethoxy) phenylhydrazone is 599 μM; and the working solution concentration of the rotenone / antimycin A mixture is 300 μM.

[0036] In step (6), the volume of oligomycin working solution added to the probe plate Port A hole is 56 μL; the volume of carbonyl cyan-4 (trifluoromethoxy) phenylhydrazone working solution added to the probe plate Port B hole is 62 μL; and the volume of rotenone / antimycin A mixture working solution added to the probe plate Port C hole is 69 μL.

[0037] The final concentration of oligomycin in each well of the cell culture plate is 30 μM; the final concentration of carbonyl cyan-4 (trifluoromethoxy) phenylhydrazone is 60 μM; and the final concentration of the rotenone / antimycin A mixture is 30 μM.

[0038] In the present application, the method for determining the cell energy metabolism of chlorella mitochondrial stress, in step (9), the volume of RPMI medium added is 400 μL. ​

[0039] In the present application, the method for determining the cell energy metabolism of Chlorella mitochondria stress is used to determine Chlorella FACHB-8, Chlorella sorokiniana FACHB-24, Chlorella sorokiniana FACHB-25, and Chlorella ellipsoidea FACHB-40.

[0040] The method for determining the cell energy metabolism of Chlorella mitochondria stress has the following beneficial effects: the method optimizes the Agilent default experimental program setting, cancels the mixing part in all reading programs; the drug concentrations of three target drugs in the mitochondrial electron transport chain are added through a large number of research and test designs, and the number of measurement cycles (5-6 times) after adding the drugs is increased to measure the oxygen consumption rate of the cells and obtain the key parameters reflecting the function of the mitochondria, so that the measurement result is more accurate. The method gives the Chlorella mitochondria stress curve, and provides a new research direction for the energy metabolism of algal cells. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The experiment example 1 is used to determine the influence of different cell densities on the basic respiration OCR value of Chlorella (FACHB-40).

[0042] Figure 2 The experiment example 2 is used to determine the influence of different Oligomycin concentrations on the ATP-related respiration OCR value of Chlorella (FACHB-40).

[0043] Figure 3 The experiment example 3 is used to determine the influence of different FCCP concentrations on the maximum respiration OCR value of Chlorella (FACHB-40).

[0044] Figure 4 The experiment example 4 is used to determine the influence of different Rotenone / Antimycin A mixture (Rot / AA) concentrations on the non-mitochondrial respiration oxygen consumption OCR value of Chlorella (FACHB-40).

[0045] Figure 5 The method for determining the cell energy metabolism of Chlorella mitochondria stress is used to determine the cell energy metabolism of Chlorella mitochondria stress.

[0046] Figure 6 The experiment example 1 is used to determine the influence of different cell densities on the basic respiration OCR value of Chlorella (FACHB-40).

[0047] Figure 7 The experiment example 1 is used to determine the influence of different cell densities on the basic respiration OCR value of Chlorella (FACHB-40).

[0048] Figure 8 The experiment example 1 is used to determine the influence of different cell densities on the basic respiration OCR value of Chlorella (FACHB-40).

[0049] Figure 9Proton leakage OCR values of different Chlorella species in Example 1.

[0050] Figure 10 Maximum respiratory OCR values of different Chlorella species in Example 1.

[0051] Figure 11 Standby respiratory capacity OCR values of different Chlorella species in Example 1.

[0052] Figure 12 Non-mitochondrial oxygen consumption OCR values of different Chlorella species in Example 1.

[0053] Figure 13 Chlorella mitochondrial stress profile graphs under different H2O2 concentrations in Example 2.

[0054] Figure 14 Basal respiratory OCR values of Chlorella under different H2O2 concentrations in Example 2.

[0055] Figure 15 ATP-related respiratory OCR values of Chlorella under different H2O2 concentrations in Example 2.

[0056] Figure 16 Proton leakage OCR values of Chlorella under different H2O2 concentrations in Example 2.

[0057] Figure 17 Maximum respiratory OCR values of Chlorella under different H2O2 concentrations in Example 2.

[0058] Figure 18 Standby respiratory capacity OCR values of Chlorella under different H2O2 concentrations in Example 2.

[0059] Figure 19 Non-mitochondrial oxygen consumption OCR values of Chlorella under different H2O2 concentrations in Example 2. DETAILED DESCRIPTION

[0060] The technical solutions of the present application will be described in detail below with reference to the accompanying drawings.

[0061] In the examples, the specific conditions not mentioned are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents, instruments and algae species used, if not mentioned, are all conventional products that can be purchased on the market.

[0062] Example 1

[0063] The method for determining the mitochondrial stress cell energy metabolism of Chlorella comprises the following steps:

[0064] (1) Preparation: 24h before the experiment, open the Agilent Cell Energy Meter XF24 software Wave, connect the controller and the instrument host successfully, and warm up to 28℃;

[0065] And add 1mL of XF Calibrator calibration solution with pH 7.4 to each well of the hydrated plate on the clean bench, and place the entire probe plate device in a 28℃ CO2-free cell incubator for 12h of hydration.

[0066] (2) During the experiment, add 50μL of Cell-Tak coating solution with a concentration of 22.4μg / mL to each well of the cell culture plate on the clean bench; after 20min of incubation at room temperature, wash each well twice with 200μL of sterile water, and wait for use.

[0067] (3) Use a flow cytometer to count Chlorella (FACHB-8), Chlorella sorokiniana (FACHB-24, FACHB-25), and Chlorella ovalis (FACHB-40), respectively, and adjust the volume of Chlorella to make the total number of Chlorella cells in 1mL of Chlorella liquid 2.0×10 7 .

[0068] (4) Take 100μL of Chlorella liquid containing a total of 2.0×10 6 Chlorella cells and centrifuge the cells at 500×g for 5min at room temperature, then incubate the cell culture plate at room temperature for 25-30min to ensure that the Chlorella is completely attached to the bottom.

[0069] (5) Use XF RPMI medium to prepare drug working solutions with a concentration of 298μM oligomycin, a concentration of 599μM carbonyl cyanide-4(trifluoromethoxy) phenylhydrazone, and a concentration of 300μM rotenone / antimycin A mixture.

[0070] (6) Take the hydrated probe plate from the 28℃ CO2-free cell incubator, add 56μL of oligomycin to the Port A well of the probe plate, add 62μL of carbonyl cyanide-4(trifluoromethoxy) phenylhydrazone to the Port B well of the probe plate, and add 69μL of rotenone / antimycin A mixture to the Port C well of the probe plate. After the instrument injects the drugs according to the program settings, the final concentration of oligomycin in each well of the cell culture plate is 30μM, the final concentration of carbonyl cyanide-4(trifluoromethoxy) phenylhydrazone is 60μM, and the final concentration of rotenone / antimycin A mixture is 30μM.

[0071] (7) Set the cell energy metabolism instrument program, open the mitochondrial stress experiment template in the software, and first set the OCR measurement for 3 cycles of basic respiration;

[0072] Then, the instrument injects oligomycin drug working solution, and measures 6 cycles;

[0073] The instrument injects carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone drug working solution, and measures 5 cycles;

[0074] The instrument injects rotenone / antimycin A mixture drug working solution, and measures 4 cycles;

[0075] Each cycle procedure is 2 min waiting and 3 min measurement.

[0076] After the program setting is completed, the probe plate and the hydration plate are placed in the instrument for probe calibration.

[0077] (9) During the waiting calibration process, 400 μL of RPMI medium is slowly added to each well of the cell culture plate, and after 1 min of standing, the Chlorella adhesion is checked under a microscope. After adhesion is completed, the Chlorella is allowed to react in the dark at room temperature for 20 min.

[0078] (10) After calibration is completed, the cell culture plate that has been treated in the dark is used to replace the hydration plate, and detection is performed.

[0079] (11) The Agilent Cell Energy Meter XF24 software Wave automatically calculates the Chlorella mitochondrial stress curve graph.

[0080] The curve trend measured for different species of Chlorella conforms to the mitochondrial stress curve graph as shown in Figure 6 , and the relevant parameter values are calculated according to the experimental data results, as shown in Table 1.

[0081] According to Figures 7-12 and the results in Table 1, the mitochondrial functions of different species of Chlorella have certain differences. The values of basal respiration, proton leakage, and non-mitochondrial oxygen consumption of FACHB-8 are the largest; and the values of ATP-related respiration, maximum respiration, and spare respiratory capacity of FACHB-25 are the largest.

[0082] Table 1 Parameter data of mitochondrial functions of different species of Chlorella

[0083]

[0084] Example 2

[0085] The method for determining the mitochondrial stress cell energy metabolism of Chlorella comprises the following steps:

[0086] (1) Preparation: 6 equal amounts of Chlorella (FACHB-40) are respectively cultured in H2O2 solutions with concentrations of 0 μM, 150 μM, 300 μM, 400 μM, 500 μM, and 600 μM for 2 days, to obtain 6 samples.

[0087] 24h before the experiment, open the Agilent Cell Energy Meter XF24 software Wave, wait for the controller to connect successfully with the instrument host, and warm up to 28℃;

[0088] And add 1mL of XF Calibrator calibration solution with pH 7.4 to each well of the hydrated plate on the clean bench, and place the entire probe plate device in a 28℃ CO2-free cell incubator for 12h of hydration.

[0089] (2) During the experiment, add 50μL of Cell-Tak coating solution with a concentration of 22.4μg / mL to each well of the cell culture plate on the clean bench; after 20min of incubation at room temperature, wash each well twice with 200μL of sterile water, and wait for use.

[0090] (3) Use a flow cytometer to count the Chlorella under different H2O2 concentration culture conditions, and adjust the volume of Chlorella to make the total number of Chlorella cells in 1mL of Chlorella liquid 2.0×10 7 .

[0091] (4) Take 100μL of Chlorella liquid containing a total of 2.0×10 6 Chlorella cells and centrifuge the cells at 500×g for 5min at room temperature, then incubate the cell culture plate at room temperature for 25-30min to ensure that the Chlorella is completely attached to the bottom.

[0092] (5) Use XF RPMI medium to prepare drug working solution of oligomycin with a concentration of 298μM, carbonyl cyanide-4(trifluoromethoxy) phenylhydrazone with a concentration of 599μM, and a mixture of rotenone / antimycin A with a concentration of 300μM.

[0093] (6) Take the hydrated probe plate from the 28℃ CO2-free cell incubator, add 56μL of oligomycin to the Port A well of the probe plate; add 62μL of carbonyl cyanide-4(trifluoromethoxy) phenylhydrazone to the Port B well of the probe plate; add 69μL of a mixture of rotenone / antimycin A to the Port C well of the probe plate, and after the instrument injects the drugs according to the program settings, the final concentration of oligomycin in each well of the cell culture plate is 30μM, the final concentration of carbonyl cyanide-4(trifluoromethoxy) phenylhydrazone is 60μM, and the final concentration of the mixture of rotenone / antimycin A is 30μM.

[0094] (7) Set the cell energy metabolism instrument program, open the mitochondrial stress experiment template in the software, and first set the OCR measurement for 3 cycles of basic respiration;

[0095] Then, the instrument injects the oligomycin drug working solution and measures for 6 cycles;

[0096] The instrument injects the carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone drug working solution, and measures 5 cycles;

[0097] The instrument injects the rotenone / antimycin A mixture drug working solution, and measures 5 cycles;

[0098] In each cycle, the procedure is: 2 min waiting, 3 min measurement.

[0099] After the program setting is completed, the probe plate and the hydration plate are placed in the instrument for probe calibration.

[0100] (9) During the waiting calibration process, 400 μL of RPMI medium is slowly added to each well of the cell culture plate, and after 1 min of standing, the Chlorella adhesion is checked under a microscope. After adhesion is completed, the Chlorella is allowed to react in the dark at room temperature for 20 min.

[0101] (10) After calibration is completed, the cell culture plate that has been treated in the dark is used to replace the hydration plate for detection.

[0102] (11) The Agilent Cell Energy Meter XF24 software Wave automatically calculates the Chlorella mitochondrial stress curve graph.

[0103] The Chlorella curve trend measured at different H2O2 concentrations conforms to the mitochondrial stress curve graph as shown in Figure 13 , and the relevant parameter values are calculated according to the experimental data results, as shown in Table 2.

[0104] According to Figures 14-19 and the results in Table 2, when the H2O2 concentration is 150 μM and 300 μM, the maximum respiration value of Chlorella increases; when the H2O2 concentration is 400 μM, the maximum respiration value of Chlorella decreases; when the H2O2 concentration is 500 μM and 600 μM, the mitochondrial function of Chlorella is impaired, the basal respiration significantly decreases, and there is no obvious mitochondrial stress curve trend, and the function parameter data cannot be calculated. The experimental results show that at low H2O2 concentration, the mitochondrial function of Chlorella is enhanced, and at high H2O2 concentration, the mitochondrial function of Chlorella is reduced or even lost.

[0105] Table 2 Parameter data of Chlorella mitochondrial function at different H2O2 concentrations

[0106]

[0107]

[0108] Note: - indicates that the mitochondrial stress experiment does not conform to the curve trend, and the data cannot be calculated

[0109] Experimental Example 1

[0110] I. Experimental purpose: test the effect of different cell densities on the basal respiration OCR value of Chlorella (FACHB-40).

[0111] II. Experimental method: combine the basal respiration OCR value with the requirement that the cell number at the bottom of the hole plate be evenly distributed, not stacked, and the plate bottom coverage rate, adjust the volume of Chlorella, set the cell density gradient of Chlorella, so that the total number of cells in 1 mL of Chlorella liquid is 4.0 x 10 6 , 8.0 x 10 6 , 1.0 x 10 7 , 2.0 x 10 7 , 4.0 x 10 7 , 8.0 x 10 7 , 1.0 x 10 8 . Take 100 μL of Chlorella liquid and add it to the cell culture plate, then the total number of Chlorella cells in each hole is 4.0 x 10 5 , 8.0 x 10 5 , 1.0 x 10 6 , 2.0 x 10 6 , 4.0 x 10 6 , 8.0 x 10 6 , 1.0 x 10 7 .

[0112] The experiment was carried out according to the steps described in Example 1.

[0113] III. Experimental results: as shown in Figure 1 . Through Figure 1 analysis, the total number of Chlorella cells in each hole is 2.0 x 10 6 , which is the best choice.

[0114] Experimental Example 2

[0115] I. Experimental purpose: test the effect of Oligomycin concentration on the ATP-related respiration OCR value of Chlorella (FACHB-40).

[0116] II. Experimental method: set the Oligomycin concentration gradient to 0, 10, 20, 30, 40, 50 μM, and carry out the experiment according to the steps described in Example 1.

[0117] III. Experimental results: as shown in Figure 2 . Through Figure 2 analysis, when the Oligomycin concentration is 30 μM, the ATP-related respiration OCR value is the largest, so the optimal concentration of Oligomycin is 30 μM.

[0118] Experimental Example 3

[0119] I. Experimental Objective: To test the effect of carbonyl cyano-4 (trifluoromethoxy)phenylhydrazone (FCCP) concentration on the ATP-related respiratory OCR value of Chlorella (FACHB-40).

[0120] II. Experimental Methods: The concentration of Oligomycin was fixed at 30 μM, and the FCCP concentration gradient was set to 0, 20, 40, 60, 80, 100, and 120 μM.

[0121] III. Experimental Results: Figure 3 As shown. (Through) Figure 3 Analysis shows that the maximum respiratory OCR value is reached at an FCCP concentration of 60 μM after drug injection, therefore the optimal concentration of FCCP is 60 μM.

[0122] Example 4

[0123] I. Experimental Objective: To test the effect of rotenone / antimycin A mixture (Rot / AA) concentration on the oxygen consumption (OCR) value of non-mitochondrial respiration in Chlorella (FACHB-40).

[0124] II. Experimental Methods: The concentration of Oligomycin was fixed at 30 μM and the concentration of FCCP was fixed at 60 μM. The Rot / AA concentration gradient was set to 0, 10, 20, 30, 40, 50, and 60 μM.

[0125] III. Experimental Results: Figure 4 As shown. (Through) Figure 4 Analysis shows that after drug injection, the non-mitochondrial oxygen consumption OCR value reaches its lowest value when the Rot / AA concentration is 30 μM. Therefore, the optimal concentration of FCCP is 30 μM.

Claims

1. A method of determining the cell energy metabolism of Chlorella mitochondria stress, characterized by, The method comprises the following steps: (1) Preparation: 24 hours before the experiment, open the Agilent Cell Energy Meter XF24 software Wave, and warm up to 25-28℃; Add 1 mL of XF Calibrant calibration solution with pH 7.4 to each well of the hydration plate, and place the entire probe plate device in a 25-28℃ CO2-free cell incubator for 12 hours of hydration; (2) Add 50-55 μL of Cell-Tak coating solution with a concentration of 22-22.5 μg / mL to each well of the cell culture plate; After 20-25 minutes of incubation at room temperature, wash each well twice with sterile water, and wait for use; (3) Counting the Chlorella by using a flow cytometer, adjusting the volume of the Chlorella so that the total number of Chlorella cells in 1 mL of Chlorella liquid is 1.5 x 10 7 -2.5 x 10 7 cells; (4) Take 100 μL of the Chlorella liquid containing 1.5 x 10 6 -2.5 x 10 6 cells and centrifuge the cells at 500 x g for 5 min at room temperature. Then, incubate the cell culture plate at room temperature for 25-30 min to ensure that the Chlorella cells are completely attached to the bottom of the plate. (5) Use the XF RPMI medium to configure the drug working solution of oligomycin, carbonyl cyanide-4(trifluoromethoxy) phenylhydrazone, and rotenone / antimycin A mixture; (6) Take the hydrated probe plate from the 25-28℃ CO2-free cell incubator, and add oligomycin to the Port A well of the probe plate; add carbonyl cyanide-4(trifluoromethoxy) phenylhydrazone to the Port B well of the probe plate; Add rotenone / antimycin A mixture to the Port C well of the probe plate; after the instrument sequentially injects the drugs according to the program setting, the final concentration of oligomycin in each well of the cell culture plate is 25-35 μM, the final concentration of carbonyl cyanide-4(trifluoromethoxy) phenylhydrazone is 55-65 μM, and the final concentration of rotenone / antimycin A mixture is 25-35 μM; (7) Set the cell energy metabolism instrument program, open the mitochondrial stress experiment template in the software, and first set the basic respiration OCR measurement for 3 cycles; Then, the instrument injects the oligomycin drug working solution, and measures for 6 cycles; The instrument injects the carbonyl cyanide-4(trifluoromethoxy) phenylhydrazone drug working solution, and measures for 5 cycles; The instrument injects the rotenone / antimycin A mixture drug working solution, and measures for 4-5 cycles; Each cycle program is 2 minutes of waiting and 3 minutes of measurement. (8) After the program setting is completed, place the drug-added probe plate and the hydration plate into the instrument for probe calibration; (9) During the waiting calibration process, add RPMI medium to each well of the cell culture plate, and after 1 minute of standing, check the adhesion of chlorella under a microscope, and after the adhesion is completed, react in the dark at room temperature for 20 minutes; (10) After the calibration is completed, replace the hydration plate with the cell culture plate that has been treated in the dark, and perform detection; (11) The Agilent Cell Energy Meter XF24 software Wave automatically calculates the chlorella mitochondrial stress curve graph.

2. The method of determining the cell energy metabolism of the Chlorella mitochondrial stress according to claim 1, characterized in that, In step (1), the temperature is raised to 28℃.

3. The method of determining the cell energy metabolism of the Chlorella mitochondrial stress according to claim 1, characterized in that, In step (2), the concentration of the Cell-Tak coating solution is 22.4 μg / mL, and the amount used is 50 μL; the amount of sterile water used is 200 μL.

4. The method of determining the mitochondrial stress cell energy metabolism of Chlorella according to claim 1, characterized in that, The total number of Chlorella cells in 1 mL in the step (3) is 2 x 10 7 The total number of Chlorella cells in 100 μL of Chlorella liquid taken in the step (4) is 2 x 10 6 The total number of Chlorella cells in 100 μL of Chlorella liquid taken in the step (4) is 2 x 10 5. The method of determining the mitochondrial stress cell energy metabolism of Chlorella according to claim 1, characterized in that, In step (5), the concentration of the oligomycin drug working solution is 298 μM; the concentration of the carbonyl cyanide-4(trifluoromethoxy) phenylhydrazone drug working solution is 599 μM; and the concentration of the rotenone / antimycin A mixture drug working solution is 300 μM; In step (6), the volume of the oligomycin drug working solution added to the Port A well of the probe plate is 56 μL; The volume of the rotenone / antimycin A mixture drug working solution added to the port B hole of the probe plate was 69 μL. The volume of the rotenone / antimycin A mixture drug working solution added to the port B hole of the probe plate was 69 μL. The final concentration of oligomycin in each hole of the cell culture plate was 30 μM; the final concentration of cyanoginoside-4 (trifluoromethoxy) phenylhydrazone was 60 μM; and the final concentration of rotenone / antimycin A mixture was 30 μM.

6. The method of determining the mitochondrial stress cell energy metabolism of Chlorella according to claim 1, characterized in that, The volume of the RPMI medium added in the step (9) was 400 μL.

7. The method of determining the mitochondrial stress cell energy metabolism of Chlorella according to claim 1, characterized in that, Chlorella vulgaris FACHB-8, Chlorella sorokiniana FACHB-24, Chlorella sorokiniana FACHB-25, Chlorella ovalis FACHB-40.

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