CN114577774B - Mitochondrial probe fluorescence intensity correction method and cell concentration detection method - Google Patents
Mitochondrial probe fluorescence intensity correction method and cell concentration detection method Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention provides a fluorescence intensity correction method and a cell concentration detection method for a mitochondrial probe. The correction method comprises the following steps: s110: obtaining cell concentration value X of standard sample, detectingFluorescence intensity value Y of the mitochondrial probe corresponding to the cell concentration value X; s120: establishing a Logit correction model according to the cell concentration value X and the fluorescence intensity value Y of the standard sample; s130: according to the Logit correction model, the true value y of the fluorescence intensity of the sample to be detected i Corrected to the fluorescence intensity correction value y k . The real value y of the fluorescence intensity by the correction method provided by the invention i And (3) correcting to ensure that the fluorescence intensity value is not influenced by the cell concentration value, and improving the detection accuracy of the mitochondrial probe.
Description
Technical Field
The invention relates to the technical field of mitochondrial probes, in particular to a fluorescence intensity correction method and a cell concentration detection method of a mitochondrial probe.
Background
Mitochondria, which are the major energy-supplying organelles in eukaryotic cells, are important areas of cellular metabolism, and are called "energy factories of cells" for their important role in energy metabolism. Mitochondria have been called important research targets due to their unique aspects (e.g., genetic material containing mtDNA, their own protein synthesis mechanisms, and tightly regulated membrane potential gradients). Meanwhile, with the research on the aspects of immune aging and the like, the effect of mitochondria is more remarkable than before.
Healthy mitochondria have a well-established electron transport chain-generated lower steric membrane potential (Δ Ψ m), which, along with the proton gradient, generates the driving force behind mitochondrial ATP synthesis. The detection of mitochondrial function can be generally evaluated in terms of the whole level or the isolated level, and the evaluation indexes include Mitochondrial Membrane Potential (MMP), Mitochondrial Mass (MM), mitochondrial oxygen consumption (ROS), mitochondrial oxidative respiratory chain function measurement, mitochondrial DNA function measurement, and the like. Among them, measurement of mitochondrial membrane potential and mitochondrial mass is the most widely used functional index at the cellular level, such as the classical JC-1 probe, which detects changes in mitochondrial membrane potential through the shift of fluorescence color, and the Mito Trackcer series probe, which utilizes the thio-reactive chloromethyl group, labeled and retained in mitochondria, detects mitochondrial mass through the fluorescence intensity value.
The mitochondrial probe mentioned in the patent of 'a fluorescent probe for simultaneously detecting mitochondrial membrane potential and quality by a flow cytometer and a synthetic method thereof' has the same function as a MitoTracker series in principle, the marking function mainly depends on the mitochondrial membrane potential, and the specificity of a probe reaction group is well reserved in mitochondria, so that the detection of the mitochondrial quality or the membrane potential is achieved. According to the Nernst equation, the membrane potential is related to the ion concentration (pH, temperature dependence), while the K in the solution is + Changes in ion concentration also affect mitochondrial membrane potential.
Therefore, when the membrane potential label design type probe is used for carrying out mitochondrial quality detection, the binding is related to the ion concentration (mainly cell concentration), and the binding area of the probe is increased along with the increase of the cell concentration, so that the fluorescence intensity value is reduced. Due to the characteristics, the same sample can present different fluorescence intensity values under different cell concentrations, so the direct detection result of the probe cannot represent the mitochondrial quality value, and how to eliminate the influence of the cell concentration on the mitochondrial fluorescence intensity value has important significance for detecting the mitochondrial quality.
Disclosure of Invention
The invention aims to solve the influence of cell concentration on the fluorescence intensity of a mitochondrial probe, and provides a method for correcting the fluorescence intensity of the mitochondrial probe, which comprises the following steps:
s110: obtaining a cell concentration value X of a standard sample, and detecting a fluorescence intensity value Y of a mitochondrial probe corresponding to the cell concentration value X;
s120: establishing a Logit correction model according to the cell concentration value X and the fluorescence intensity value Y of the standard sample;
s130: according to the Logit correction model, the true value y of the fluorescence intensity of the sample to be detected i Corrected to the fluorescence intensity correction value y k 。
Since the cell membrane itself also has a membrane potential, the cell concentration is inversely related to the concentration of the mitochondrial probe, which is reflected in the fluorescence intensity values on the flow cytometer. In the present invention, a Logit correction model is established by studying the correlation between the cell concentration value X and the fluorescence intensity value Y of the standard sample. For example, the Logit correction model may be obtained by: preparing standard samples with different cell concentration gradients, carrying out flow cytometry circle on target cells, analyzing mitochondrial probe fluorescence intensity values Y of the target cells corresponding to different cell concentrations, and analyzing the relationship between the cell concentration value X and the fluorescence intensity value Y. Then, according to the Logit correction model, the true fluorescence intensity value y of the sample to be detected i Corrections were made to exclude the effect of cell concentration.
Further, the Logit correction model is obtained by:
s121: determining a cell concentration processing value according to the cell concentration value X;
s122: determining a fluorescence intensity processing value according to the fluorescence intensity value Y;
s123: the Logit correction model is obtained by linear regression of the cell concentration treatment value and the fluorescence intensity treatment value.
In the invention, the cell concentration processing value and the fluorescence intensity processing value show linear changes, and a regression equation can be obtained through linear regression, namely the Logit correction model provided by the invention. In the Logit correction model, the fluorescence intensity treatment value varies with the change in the cell concentration treatment value, as is the case with the standard sample, and as is the case with the unknown sample to be tested, so that the other value can be determined from the Logit correction model as long as one of the fluorescence intensity treatment value and the cell concentration treatment value is known.
Further, the cell concentration processing value is a logarithmic value of the cell concentration value X; the fluorescence intensity processing value is a logarithmic value of the fluorescence intensity value X; the Logit correction model is: log (y) = nlog (x) + a; where n is a constant and a is a constant.
In the invention, experimental research shows that the fluorescence intensity value Y of the mitochondrial probe and the cell concentration value X are in a power exponent correlation coefficient, so that the cell concentration value X and the cell mitochondrial detection fluorescence intensity value Y are subjected to logarithmic processing, and linear regression can be observed by performing correlation analysis on the processed data; the Logit correction model is also: log (y) = nlog (x) + a. Where n and a are both constants, which can be determined by analysis of a standard sample. In particular, all log functions in the present invention are based on 10.
Further, step S130 includes the steps of:
s131: obtaining the true value x of the cell concentration of the sample to be detected i Detecting the true value x of the cell concentration i Corresponding true value y of fluorescence intensity i ；
S132: according to the Logit correction model, the true value y of the fluorescence intensity is calculated i Correcting to the same cell concentration correction value x k Corresponding fluorescence intensity correction value y k 。
In the invention, the true value y of the fluorescence intensity of the sample to be detected can be realized based on the established Logit correction model i And (4) correcting. Aiming at the sample to be detected, the true value x of the cell concentration can be obtained i And the corresponding true value y of the fluorescence intensity i (ii) a To eliminate the influence of the cell concentration, the true value y of the fluorescence intensity may be i Correcting to the same corrected value x of the cell concentration k Corresponding fluorescence intensity correction value y k . Wherein the cell concentration correction value x k Different values may be assigned according to the actual requirements, which values are constant in a specific embodiment.
Further, the true value x of the cell concentration i And the true value y of the fluorescence intensity i The method meets the Logit correction model: log (y) i ）=nlog（x i ）+a；
Correction value x for cell concentration k And the fluorescence intensity correction value y k The method meets the Logit correction model: log (y) k ）=nlog（x k ）+a。
In the invention, the true value and the correction value both meet the Logit correction model, so that the algorithm can be simplified according to the commonality of the true value and the correction value in the correction process, and the subsequent data processing is convenient. The derivation process is as follows:
further, the fluorescence intensity correction value y k From the actual value x of the cell concentration i True value y of fluorescence intensity i Cell concentration correction value x k And the constant n determines that:
in the present invention, the constant a varies greatly for different samples and detection means, so that the constant a is eliminated by inference and only the true value x of the cell concentration is used i True value y of fluorescence intensity i Cell concentration correction value x k The corrected value y of the fluorescence intensity can be determined by the constant n k 。
Further, the value range of n is-0.8 to-0.5.
In the invention, the value range of n will fluctuate with the setting schemes of different instruments, so that the detection means and the detection environment should be kept consistent when the sample to be detected is detected. The value range fluctuation of the constant n is very small relative to the constant a; this n value was used in a NovoCyte flow cytometer, CD3+% cell population.
Further, the cell concentration value X is a leukocyte concentration value.
In the invention, a leukocyte concentration value is used in the process of establishing the Logit correction model; among leukocytes, there are mainly lymphocyte population (Lym%), monocyte population and granulocyte population, and although lymphocyte population is mainly studied, a Logit model of fluorescence intensity values of human whole blood samples and cell mitochondria is established as an example:
1. labeling the lymphocyte population with CD4, CD8, CD62L and CD45RA, wherein the CD4+ T lymphocyte population is the white blood cell population in the FSC/SSC circle (absolute count is L true test value/. mu.l), in the lymphocyte population (L true test value x Lym%), the CD4+ T cell population is read at a ratio of a%, and similarly the CD45RA + CD62L + cell population (na dove CD4+ T cell population) is read in the CD4+ T cell population at a ratio of B%;
2. according to the above ratio, the absolute cell number of CD4+ T cell population can be found to be L true test value ×% Lym% ×% a%/μ L, and the absolute cell number of na meive CD4+ T cell population can be found to be L true test value ×% Lym% ×% a%/B% μ L;
3. as the concentration of the cells is reduced in the process of dilution, the cell groups are not changed, namely Lym%, A% and B% are not changed.
Therefore, the leukocyte concentration value can be used as it is without conversion.
In another aspect, the present invention further provides a method for detecting cell concentration based on a mitochondrial probe, comprising the following steps:
s210: obtaining a sample to be detected marked by the mitochondrial probe, and detecting the fluorescence intensity value y of the mitochondrial probe n ；
S220: determining the cell concentration value x of the sample to be detected according to the Logit correction model n ；
The Logit correction model is obtained by establishing the correction method.
In the invention, the Logit correction model reflects the relationship between the cell concentration value and the fluorescence intensity value, so that the cell concentration value of a sample can be calculated when the fluorescence intensity value of the sample to be detected is obtained based on the model.
Further, cell concentration value x n And fluorescence intensity value y n Satisfies log (y) n ）=nlog（x n ) + a; where n is a constant and a is a constant.
Drawings
Fig. 1 shows the data processing results provided in embodiment 1 of the present invention.
FIG. 2 is a schematic representation of y according to some embodiments of the present invention k The schematic is derived.
FIG. 3 is a comparison between the fluorescence intensity of mitochondria in example 1 of the present invention before and after calibration.
Fig. 4 is a Logit correction model established in embodiment 2 of the present invention.
FIG. 5 shows the target cell population in FSC/SSC cycles of example 3 of the present invention.
Fig. 6 is a Logit correction model established in embodiment 3 of the present invention.
FIG. 7 shows the CD4+ CD 8-cell population at the 4 th round of the present invention.
FIG. 8 shows the CD4-CD8+ cell population at the 4 th round of the present invention.
FIG. 9 is a Logit correction model for CD4+ T cells and Na meive CD4+ T cells of example 4 of the invention.
FIG. 10 is a Logit correction model for CD8+ T cells and Na meive CD8+ T cells of example 4 of the invention.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Firstly, establishing a Logit correction model
1. A peripheral blood sample was diluted to 7 concentration points, 3.4X 10, respectively, in accordance with the leukocyte concentration 3 ，5.1×10 3 ，7.6×10 3 ，11.4×10 3 ，17.1×10 3 ，25.6×10 3 unit/. mu.L, selection of the target cell population nameive CD4+ T (CD 4+ CD45RA + CD62L +) analysis of mitochondrial fluorescence intensity values for this population, with the raw data results shown below in Table 1:
TABLE 1
2. Establishing a Logit correction model according to the original data: log (y) = nlog (x) + a; the results obtained by linear regression are shown in FIG. 1. Wherein a is a constant, and the n value obtained by establishing the model is as follows: n (CD3) = -0.8 to-0.5.
3. Deducing a fluorescence intensity correction value y of a sample to be detected according to a Logit correction model k ： 
Wherein, y k Is a fluorescence intensity correction value; x is the number of i The actual value of the cell concentration; y is i The real value of the fluorescence intensity; x is the number of k Is a cell concentration correction value; n is a constant. The derivation diagram is shown in FIG. 2
Secondly, calibrating the original data and the cell concentration correction value x in the table 1 through a Logit correction model k Take 5X 10 3 unit/. mu.L; the results are shown in Table 2. The comparison before and after correction is shown in fig. 3.
TABLE 2
Example 2
Firstly, establishing a Logit correction model
Using CD3 FITC and a mitochondrial probe to respectively dye and mark three samples as sample 1, sample 2 and sample 3; the results of cell counting using absolute counting microspheres in a flow cytometer kit for sample 1 are shown in table 3.
TABLE 3
Referring to fig. 4, a Logit correction model is established according to the original data: log (y) = -0.6964log (x) + 5.5849.
Secondly, calculating the cell concentration through a Logit correction model
Diluting the sample 2 for 2 points, and marking as an unknown concentration point C11 and an unknown concentration point C12; diluting the sample 3 for 2 points, and marking as an unknown concentration point C21 and an unknown concentration point C22; the absolute count method of the reference sample 1 detects the absolute count value x of the cells at the four unknown concentration points.
TABLE 4
X in the table n The cell concentration value is calculated by a Logit correction model, and x is the absolute count of the cell and is detected by a flow cytometer.
Example 3 Logit calibration model for establishing cell concentration value X and fluorescence intensity value Y of standard cell strain
1. Cell sample preparation
1.1 the cell laboratory is disinfected conventionally, the ultraviolet irradiation is carried out for more than 30min, and the super clean bench is opened for ventilation for 10 min.
1.2 cell culture Medium (1640+10% FBS) was preheated at 37 ℃ and sterilized by wiping with 75% alcohol before being transferred to a cell ultra-clean bench.
1.3 taking out the cell cryopreservation tube from the liquid nitrogen tank, immediately placing the tube in a water bath at 37 ℃ and quickly shaking the cell cryopreservation tube to quickly melt the tube.
1.4 transfer the cells in the cryopreserved tubes to a 15mL centrifuge tube in a clean bench, add 3mL of fresh cell culture medium and centrifuge at 800 rpm for 5 min.
1.5 in cell culture flasks (25 cm) during the waiting time for centrifugation 2 ) To this was added 5ml of fresh complete cell culture medium.
1.6 after centrifugation, the supernatant was discarded, 1mL of fresh medium was added to the cell pellet and gently blown down to mix well, and the cells were removed and added to a culture flask. Gently mixing all the materials, placing in a cell culture box at 37 deg.C and 5% CO 2 And (5) culturing.
1.7 when the density of cultured cells reaches 2.0X 10 6 At approximately/mL, a cell staining marker experiment was performed.
1.8 gradient dilution of cultured cells to 7 concentration points, 0.5X 10 3 ，1×10 3 ，5×10 3 ，10×10 3 ，15×10 3 ，20×10 3 ，30×10 3 And (4) taking the cell strain samples with different concentrations, and adding MitoDye with the concentration range of 0.025-0.1 mu g/test into the cell strain samples with different concentrations.
1.9 incubate in the dark at 37 ℃ for 30 minutes.
1.10 detecting on an Agilent NovoCyte flow cytometer, wherein the acquisition speed is medium (35-60 muL/min), and the acquisition graph template is shown in figure 5;
2. establishing a Logit correction model
2.1 median fluorescence intensity values in their MitoDye (APC channel) were counted by centering the target cell population in FSC/SSC circles.
2.2 cell concentration values X and their MitoDye median fluorescence intensity values Y of the standard cell lines are summarized, and the data results are shown in Table 5.
TABLE 5
2.3 referring to fig. 6, a Logit correction model is established according to the experimental result: log (y) = -0.8818 log (x) + 6.4716.
3. Correcting the fluorescence intensity value of the sample to be detected according to the Logit correction model;
correction value of fluorescence intensity
Wherein x is i The actual value of the cell concentration; y is i The real value of the fluorescence intensity; x is the number of k The cell concentration correction value can be assigned according to specific requirements; n is a constant, and in this embodiment n is-0.8818. x is the number of i 、y i 、x k N can be determined, and thus the fluorescence intensity correction value y can be calculated k 。
Example 4 Logit model for establishing cell concentration value X and cell mitochondria fluorescence intensity value Y of human whole blood sample
1. Sample preparation
1.1 at room temperature, taking a proper volume of antibody detection reagent into a marked flow tube;
1.2 mu.L of well-mixed human peripheral blood samples (1X 10) of anticoagulant of different concentrations 3 ，2.5×10 3 ，5×10 3 ，10×10 3 ，20×10 3 ，40×10 3 ) Adding into the bottom of the test tube;
note that: the method avoids the problem that the blood sample on the tube wall cannot be dyed when touching the tube wall in the operation, which can affect the experimental result; and reverse sample loading is needed when a human peripheral blood sample is removed.
1.3 gently oscillating on a vortex mixer for 5 seconds, standing and incubating for 15 minutes at room temperature in a dark place;
1.4 adding 2mL of hemolysin working solution placed at room temperature;
1.5 gently shaking on a vortex mixer for 5 seconds, standing and incubating for 15 minutes at room temperature in a dark place;
1.6 after incubation, slightly mixing, putting 200 mu L of the mixture into a new flow tube, and counting on a machine;
1.7 centrifuging the rest sample at room temperature for 5min at 300 g;
1.8 discarding the supernatant, adding 200. mu.L PBS for resuspension;
1.9 taking 200 mu L of the resuspended sample, adding the sample into the MitoDye probe hole, gently swirling and mixing the mixture evenly, and incubating the mixture for 30min at 37 ℃ in a dark place under an electric heating constant temperature incubator;
1.10 detecting on Agilent NovoCyte flow cytometer, collecting the pattern template with medium speed (35-60 μ L/min), as shown in figure 7 and figure 8.
Logit correction model establishment
2.1 use FSC/SSC circle target cell population to circle CD4+ CD 8-and CD4-CD8+ cell populations in CD4 vs CD8 scatter plot, and to circle CD4+ T cells, Na-meive CD4+ and CD8+ T cells, Na-meive CD8+ T cells according to CD62L vs CD45RA scatter plot. See fig. 7 and 8.
2.2 Total sample concentration values and their MitoDye median fluorescence intensity values
2.2.1 data for CD4+ and Na meive CD4+ T cells the results are shown in Table 6:
TABLE 6
Referring to fig. 9, a Logit correction model is established;
CD4+ T cells: log (Y) 1 ） = -0.6779 log（X）+5.8252 ；
A meive CD4+ T cell: log (Y) 2 ）= -0.7805 log（X）+6.1962 。
2.2.2 data for CD8+ and Na meive CD8+ T cells the results are shown in Table 7:
TABLE 7
Referring to fig. 10, a Logit correction model is established;
CD8+ T cells: log (Y) 3 ） = -0.5078 log（X）+5.6172 ；
A meive CD8+ T cell: log (Y) 4 ）= -0.6927 log（X）+6.9819 。
3. Correcting the fluorescence intensity value of the sample to be detected according to the Logit correction model;
correction value of fluorescence intensity
Wherein x is i The actual value of the cell concentration; y is i The real value of the fluorescence intensity; x is the number of k The cell concentration correction value can be assigned according to specific requirements; n is a constant. x is the number of i 、y i 、x k N can be determined, and thus the fluorescence intensity correction value y can be calculated k 。
It is worth mentioning that with respect to x i The mathematical derivation of whether the total amount of cells, i.e. total white blood cells, is used in the model building process, but is the target cell concentration in the calibration application, will have an effect on subsequent calibrations is as follows:
among leukocytes, lymphocyte population (Lym%), monocyte population and granulocyte population are mainly classified, and although lymphocyte population is mainly studied, the human whole blood sample and the logit model of the fluorescence intensity values of cell mitochondria are used as an example:
1. labeling the lymphocyte population with CD4, CD8, CD62L and CD45RA, wherein the CD4+ T lymphocyte population is the white blood cell population in the FSC/SSC circle (absolute count is L true test value/. mu.l), in the lymphocyte population (L true test value x Lym%), the CD4+ T cell population is read at a ratio of a%, and similarly the CD45RA + CD62L + cell population (na dove CD4+ T cell population) is read in the CD4+ T cell population at a ratio of B%;
2. according to the above ratio, the absolute cell number of CD4+ T cell population can be found to be L true test value ×% Lym% ×% a%/μ L, and the absolute cell number of na meive CD4+ T cell population can be found to be L true test value ×% Lym% ×% a%/B% μ L;
3. as the concentration of the cells is reduced in the process of dilution, the cell groups are not changed, namely Lym%, A% and B% are not changed.
The mathematical formula was derived below using CD4+ T cell population (true test values L% Lym% a% cells/μ L),
i.e. x i True test values for L% Lym% a% cells/μ L,
x k the values are theoretically tested L% Lym% a% per μ L.
See fig. 2, where:
x i CD4+ T cell concentration (unit: unit X103/. mu.L) for real flow cytometry;
y i true determination of CD4+ T cell concentration for flow cytometry
(ii) a fluorescence intensity value of;
x k theoretical CD4+ T cell concentration (unit: unit/. mu.L)
y k Fluorescence intensity values detected by flow cytometry at theoretical CD4+ T cell concentrations;
the data formula is derived as follows:
according to x i And y i And a theoretical concentration x k To derive y k Value of
Carry over i.e. x i The true test values for L Lym%. A% per μ L, x k Theoretical test value of L + Lym% + a% per μ L;
dividing Lym% and A% to obtain
Thus, the target cell group y k The fluorescence intensity value is related to the total number of the leukocyte population, so that the n value obtained in the modeling process can be derived and calculated by using the absolute count value of the leukocyte population.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (5)
1. A method for correcting fluorescence intensity of a mitochondrial probe is characterized by comprising the following steps:
s110: obtaining a cell concentration value X of a standard sample, and detecting a fluorescence intensity value Y of a mitochondrial probe corresponding to the cell concentration value X;
s120: establishing a Logit correction model according to the cell concentration value X and the fluorescence intensity value Y;
s130: according to the Logit correction model, the true fluorescence intensity value y of the sample to be detected i Corrected to the fluorescence intensity correction value y k ；
The Logit correction model is obtained by the following method:
s121: determining a cell concentration processing value according to the cell concentration value X;
s122: determining a fluorescence intensity processing value according to the fluorescence intensity value Y;
s123: the Logit correction model is obtained by linear regression of the cell concentration processing value and the fluorescence intensity processing value;
the cell concentration processing value is a logarithmic value of the cell concentration value X;
the fluorescence intensity processing value is a logarithmic value of the fluorescence intensity value Y;
the Logit correction model is as follows: log (y) = nlog (x) + a;
wherein n is a constant and a is a constant;
step S130 includes the steps of:
s131: obtaining the true value x of the cell concentration of the sample to be detected i Detecting the true value x of the cell concentration i Corresponding true value y of fluorescence intensity i ；
S132: according to whatThe Logit correction model is used for calculating the true value y of the fluorescence intensity i Correcting to the same cell concentration correction value x k Corresponding fluorescence intensity correction value y k ；
True value x of the cell concentration i And the true value y of the fluorescence intensity i Satisfying the Logit correction model: log (y) i ）=nlog（x i ）+a；
The cell concentration correction value x k And the fluorescence intensity correction value y k Satisfying the Logit correction model: log (y) k ）=nlog（x k ）+a；
The fluorescence intensity correction value y k From the actual value x of the cell concentration i True value y of the fluorescence intensity i The cell concentration correction value x k And the constant n is determined to result in:
2. the method for correcting the fluorescence intensity of a mitochondrial probe according to claim 1, wherein the value of n ranges from-0.8 to-0.5.
3. The method of claim 1, wherein the cell concentration value X is a leukocyte concentration value.
4. A cell concentration detection method based on a mitochondrial probe is characterized by comprising the following steps:
s210: obtaining a sample to be detected marked by a mitochondrial probe, and detecting the fluorescence intensity value y of the mitochondrial probe n ；
S220: determining the cell concentration value x of the sample to be detected according to a Logit correction model n ；
Wherein the Logit correction model is established by the correction method provided in any one of claims 1 to 3.
5. The method of claim 4, wherein the cell concentration value x is a cell concentration value n And the fluorescence intensity value y n Satisfies log (y) n ）=nlog（x n ）+a；
Where n is a constant and a is a constant.
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| CN101545913A (en) * | 2008-03-25 | 2009-09-30 | 北京科美东雅生物技术有限公司 | Chemoluminescence immunoassay measuring kit and preparation method thereof for triiodothyronine magnetic particles |
| CN101788484B (en) * | 2010-02-23 | 2011-08-24 | 中国人民解放军第四军医大学 | Chlorine imaging method of central nervous system based on laser confocal scanning microscope system |
| JP5772425B2 (en) * | 2011-09-13 | 2015-09-02 | ソニー株式会社 | Fine particle measuring device |
| CN103411942A (en) * | 2013-08-22 | 2013-11-27 | 贵州大学 | Fluorescent probe method for detecting Cu<2+> and cell imaging |
| CN109946451B (en) * | 2019-04-16 | 2022-05-20 | 浙江诺迦生物科技有限公司 | Detection method and detection kit for cannabinoids active substances based on concentration change effect of intracellular free calcium ions |
| CN111011694B (en) * | 2019-12-30 | 2023-03-28 | 江南大学 | Preparation method of plant essential oil emulsion for inhibiting growth of fusarium graminearum |
| CN111190004B (en) * | 2020-01-10 | 2023-11-10 | 上海泰辉生物科技有限公司 | Instant detection system of immunochromatography test strip |
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