Compound dye for cell fluorescence staining in complex matrix

Abstract

Disclosed in the present invention is a compound dye for cell fluorescence staining in a complex matrix. The formula of the compound dye, based on a total volume of 100 mL, comprises: a buffer system with a pH of 6.0-8.0; a fluorescent dye which is Sybr Gold at a concentration of 2× to 40× or 2-80 mg of propidium iodide; 0.1-10 mL of triton X-100; 0-24 mL of glycerol; 0-800 mg of EDTA-disodium; and a polyamidoamine dendrimer. The two preferred compound fluorescent dye formulas of kits of the present invention can both achieve efficient staining of cells in a complex matrix and accurate counting of the cells, and the dye has excellent fluorescent visualization capability. Compared with other dyes, the compound dye has a higher staining rate (staining can be performed within 30 seconds) and a stable staining effect, and is not prone to fluorescence quenching under long-term illumination.

Description

A composite dye for fluorescent staining of cells in complex matrices Technical Field

[0001] The invention belongs to the technical field of cell fluorescence staining, and particularly relates to a compound dye for fluorescence staining of cells in a complex matrix. Background Art

[0002] Cell staining is a discipline that combines cytology and chemistry. Based on cell morphology, it utilizes the principles of chemical reactions to perform qualitative, positional, and semi-quantitative analysis of various chemical components within cells (such as enzymes, esters, sugars, metal elements, proteins, and nucleic acids). Fluorescent cell staining is particularly important for determining the number of cells within complex matrices.

[0003] Typically, cell counting in simple matrices (such as phosphate buffer systems) can be achieved using a hemocytometer. The principle is to use a special glass counting plate to perform statistical counting of cells under a microscope.

[0004] However, when the sample matrix is ​​complex (such as milk, whole blood, etc.), particles such as protein and fat in the matrix will affect the cells observed in the microscope field of view, thereby causing deviations in the test results.

[0005] Therefore, to count cells in complex matrices, it is usually necessary to specifically stain the cells with fluorescent dyes, and then count and analyze them using a fluorescence microscope. The advantage of this method is that the target cells show a fluorescent signal after fluorescent staining, while non-target particles (proteins, fat particles, etc.) have no signal or the signal is almost negligible. Therefore, reducing non-target signals can reduce their impact on the test results, making the cell count results more accurate.

[0006] However, existing cell fluorescent dye systems (such as ethidium bromide and thiazole orange) often have disadvantages such as low staining intensity, low staining efficiency (staining rate <70% within 10 minutes), slow staining rate (usually staining time >30 minutes), unstable fluorescent dyes (prone to fluorescence quenching), and high toxicity, which can lead to problems such as low cell counting results, complicated staining operations, and environmental pollution.

[0007] Currently, one of the better commercial fluorescent dyes is Sofia Green, which can stably stain somatic cells in milk, but the required staining time is still too long (about 3 minutes). In addition, when the fat content in the milk matrix is ​​too high (>5%), the sample needs to be diluted with water at a ratio of 1:1 (v:v) or even a larger multiple, which makes the operation process complicated and increases the overall detection time.

[0008] In addition, in order to achieve the effect of accurately counting cells in complex matrices, lengthy separation methods (such as membrane separation, gradient centrifugation, chromatographic column separation, electrophoresis separation, etc.) are usually required to remove the interfering matrix, and then the cells are stained and counted for analysis, which greatly increases the workload of staining and the overall operation time. This complex and time-consuming separation method makes it more difficult to accurately measure some samples that are more time-sensitive. For example, in the detection of somatic cell counts in milk, an increase in the detection time will cause a decline in the quality of the milk sample, thereby causing somatic cell aggregation. The number of somatic cells in milk is an important indicator reflecting the safety and quality of milk. A high somatic cell count usually means the presence of mastitis or other udder diseases, which will lead to a decline in milk quality and may also lead to the elimination of sick cows, thereby affecting milk production and economic benefits. Therefore, timely and effective monitoring of changes in the number of somatic cells in milk is crucial to ensuring the health of dairy cows and the quality of milk.

[0009] To address the above issues, it is necessary to develop a fluorescent staining compound and kit with good and rapid staining effect to achieve rapid and stable staining of cells in complex systems, so as to achieve the purpose of efficiently counting cells in complex matrices. Summary of the Invention

[0010] In view of the above-mentioned deficiencies in the prior art, the present invention provides a compound dye for fluorescent staining of cells in complex matrices, aiming to achieve rapid and stable cell staining to meet the purpose of accurate cell counting in complex matrices.

[0011] The present invention first provides a compound dye for fluorescent staining of cells in a complex matrix, the formula of which comprises, based on a total volume of 100 mL:

[0012] Buffer system with pH of 6.0 to 8.0;

[0013] Fluorescent dye, wherein the fluorescent dye is Sybr Gold with a final concentration of 2× to 40× or propidium iodide 2 to 80 mg;

[0014] Triton X-100 0.1-10 mL;

[0015] Glycerol 0-24 mL;

[0016] EDTA-disodium 0~800mg.

[0017] The fluorescent dye Sybr Gold was purchased from Beijing Langbolid Trading Co., Ltd., product number CG002-500 μL, 500 μL / bottle, the stock solution concentration was 10000×, diluted 10000 times to 1×, diluted 5000 times to 2×, and diluted 250 times to 40×.

[0018] Preferably, the composite dye further comprises a polyamidoamine dendrimer at a final concentration of 0 to 1 nM. Currently, polyamidoamine dendrimers (PAMAM) are available in generations 0 to 10 (G0-G10.0). Any generation can be used in this application, with generation G4.0 being preferred. The final concentration of the polyamidoamine dendrimer is preferably 1 nM.

[0019] Preferably, the buffer system is a phosphate buffer system, a Tris-HCl buffer system or a HEPES buffer system;

[0020] And / or, the buffer system contains sodium chloride for maintaining cell osmotic pressure. Generally, the sodium chloride concentration can be based on the concentration of physiological saline, that is, 0.9 g of sodium chloride is added to 100 mL of total volume.

[0021] "And / Or" means that you can choose "and" or "or". When you choose "and", both conditions need to be met. When you choose "or", you can choose only one of them.

[0022] Further preferably, the buffer system is a phosphate buffer system; the pH is 7.4; and the total volume of the compound dye is 100 mL, comprising 22.8 mg of sodium dihydrogen phosphate, 115 mg of sodium dihydrogen phosphate, and 0.9 g of sodium chloride.

[0023] Preferably, the fluorescent dye is Sybr Gold 8× to 16× or propidium iodide 10 to 40 mg.

[0024] Further preferably, the fluorescent dye is Sybr Gold 12× or propidium iodide 20 mg.

[0025] Preferably, per 100 mL of total volume, the volume of Triton X-100 is 1-5 mL; glycerol is 5-16 mL; and EDTA-disodium is 200-600 mg.

[0026] More preferably, per 100 mL of total volume, 10 mL of glycerol; 400 mg of EDTA-disodium;

[0027] When the fluorescent dye is Sybr Gold, the volume of Triton X-100 is 5 mL; when the fluorescent dye is propidium iodide, the volume of Triton X-100 is 1 mL.

[0028] In order to prevent the product from deteriorating and to preserve it for a long time, the compound dye for fluorescent staining of cells in a complex matrix preferably also includes a preservative. The preservative can be a commonly used preservative of the type and amount, for example, 30 mg of potassium sorbate can be added to 100 mL of the total volume.

[0029] The present invention further provides a kit for fluorescent staining of cells in complex matrices, comprising the compound dye.

[0030] The present invention also provides a cell fluorescence staining method comprising the following steps: mixing the compound dye with a cell sample to be tested, and then observing the sample under a fluorescence microscope. Generally, the compound dye and the cell sample to be tested can be mixed in a volume ratio of 1:1. The mixing can be performed by vortexing the mixture for rapid and thorough mixing. The vortexing time can be 10 seconds.

[0031] The cell sample to be tested is preferably milk or blood. Although the matrix components in milk or blood samples are complex, the compound dye of the present invention is still applicable.

[0032] The functions of the components of the composite dye for fluorescent staining of cells in complex matrices of the present invention are as follows:

[0033] Sybr Gold or ethidium iodide is the main dye component and plays a major role. Sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride act as pH buffers, acting as buffer solutions and maintaining the osmotic pressure of cells. Triton X-100 acts as a surfactant, enhancing the membrane permeability and the staining effect. Glycerol allows stained cells to settle quickly during observation, achieving good observation and counting in a short period of time. Disodium ethylenediaminetetraacetic acid (EDTA-disodium) is used to reduce the aggregation rate between cells to achieve accurate counting. Polyamide-amine dendrimers (PAMAM) are nano-surfactants that use their surface electrical properties to achieve cell wrapping to enhance cell dispersion and prevent the stained cell nuclei from dispersing. Furthermore, when flowing in subsequent microfluidic pipelines and other flow paths, they can play a role in surface antifouling, thereby enhancing the reusability of the pipeline. Potassium sorbate acts as a preservative, preventing the sample from deteriorating.

[0034] Beneficial effects of the present invention:

[0035] (1) The two preferred compound fluorescent dye formulas of the kit of the present invention can both achieve efficient staining of cells in complex matrices and accurate counting of their number, and the dyes have excellent fluorescence color development ability.

[0036] (2) Compared with other dyes, this composite dye has a fast dyeing rate (dyeing can be completed within 30 seconds) and is stable. It is not prone to fluorescence quenching even under long-term illumination. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 shows the test results of the effects of major fluorescent dyes on the number of somatic cells stained in milk at different staining times. The lowercase letters a and b in the figure represent statistical significance analysis. The same letters represent p>0.05, which means there is no significant difference; different letters represent p<0.05, which means there is a significant difference. In addition, in the significance analysis, only the same group analysis is performed, and no analysis between different groups is performed. The following figures are equivalent.

[0038] Figure 2 shows the effects of milk somatic cell samples stained with different dyes after continuous exposure to excitation light for 30 seconds, 5 minutes, and 10 minutes.

[0039] FIG3 is a graph showing the detection results of changes in cell number observed after staining with two dyes at different concentrations.

[0040] FIG4 is a graph showing the effect of surfactant concentration on cells stained with different dyes.

[0041] FIG5 is a graph showing the effect of pH on dye chromosome cells.

[0042] FIG6 is a graph showing the effect of temperature on dye chromosome cells.

[0043] FIG7 shows the effect of EDTA-disodium salt on the aggregation rate of somatic cells and the effect of glycerol concentration on the sedimentation time of somatic cells.

[0044] Figure 8 shows the results of testing the effect of PAMAM addition on dyes, where (A) and (B) respectively represent the 12× Sybr Gold compound dye without and with the addition of a polyamidoamine dendrimer; (C) and (D) respectively represent the 200 μg / mL propidium iodide compound dye without and with the addition of a polyamidoamine dendrimer; and (E) and (F) respectively represent the adhesion of the polyamidoamine dendrimer to the inner wall of a microfluidic channel with and without the addition of the dye.

[0045] FIG9 is a graph showing the test results of the effect of fat content in milk on the dye staining effect on cells.

[0046] FIG10 is a graph showing the test results of the effect of lactose content in milk on dye-stained cells.

[0047] FIG11 is a graph showing the test results of the effects of different glucose concentrations on dye-stained cells in a serum environment simulated by bovine serum albumin.

[0048] FIG12 is a graph showing the detection results of the effects of different protein contents on dye-stained cells. DETAILED DESCRIPTION

[0049] The optimal compound formulation of the two cell fluorescence kits is as follows:

[0050] S1 compound: 120 μL of 10000× Sybr Gold (the main fluorescent dye, purchased from Beijing Langbolid Trading Co., Ltd., product number CG002-500uL, 500 μL / bottle), 22.8 mg of sodium dihydrogen phosphate, 115 mg of sodium dihydrogen phosphate, 0.9 g of sodium chloride, 5 mL of Triton X-100, 10 mL of glycerol, 400 mg of ethylenediaminetetraacetic acid disodium salt (EDTA-disodium), 1 mL of polyamidoamine dendrimer (PAMAM) solution (prepare 0.1 μM solution first, take 14.2 mg Add the original PAMAM solution (ethylenediamine core, generation 4.0, containing 10 wt% of the original solution) to 1 L of ultrapure water; the final concentration in the compound is 1 nM. ) and 30 mg of potassium sorbate to a 100 mL volumetric flask with ultrapure water and mix thoroughly. (The excitation wavelength of the fluorescent dye in this formula is approximately 495 nm, and the emission wavelength is approximately 537 nm.)

[0051] S2 compound: 20 mg of propidium iodide (the main fluorescent dye), 22.8 mg of sodium dihydrogen phosphate, 115 mg of sodium dihydrogen phosphate, 0.9 g of sodium chloride, 1 mL of Triton X-100, 10 mL of glycerol, 400 mg of ethylenediaminetetraacetic acid disodium salt (EDTA-disodium), 1 mL of polyamidoamine dendrimer (PAMAM) solution (prepare a 0.1 μM solution by dissolving 14.2 mg of PAMAM stock solution (ethylenediamine core, generation 4.0, containing 10 wt%) in 1 L of ultrapure water; the final concentration in the compound is 1 nM), and 30 mg of potassium sorbate are brought to a 100 mL volumetric flask with ultrapure water and mixed thoroughly (the excitation wavelength of the fluorescent dye in this formula is approximately 535 nm, and the emission wavelength is approximately 617 nm).

[0052] The difference between the S1 compound and the S2 compound lies in the different amounts of the main fluorescent dye and surfactant used. The other ingredients of the formula are the same, specifically: S1 contains 120 μL Sybr Gold and 5 mL Triton X-100, and S2 contains 20 mg propidium iodide and 1 mL Triton X-100.

[0053] Here’s how to use it:

[0054] When in use, the prepared compound dye is mixed with the cell sample to be tested at a ratio of 1:1 (v / v), shaken on a vortex shaker for 10 seconds to fully mix, and the sample is dropped onto a standard card (or slide, cell counting plate) and observed under a fluorescence microscope.

[0055] Example 1: Screening of major fluorescent dyes

[0056] To identify the primary fluorescent dye with the best performance and effectiveness (i.e., fast staining rate and high number of stained cells), we used four different cell dyes (Sybr Gold, propidium iodide, Sybr Green, and thiazole orange) to stain somatic cells in a complex matrix (milk) and calculated the average number of cells observed in the microscopic field after staining. Specifically, a 50 μL + 50 μL volume of 2× Sybr Gold, 100 μg / mL propidium iodide, 2× Sybr Green, 2 μM thiazole orange, and 465 cells / μL milk standard sample were added to a test tube and shaken for 10 seconds. 8 μL of the mixed sample was then dropped onto a standard card and counted under an inverted fluorescence microscope.

[0057] As shown in Figure 1, Sybr Gold dye achieved a maximum staining rate of approximately 100 cells within 30 seconds. The observed cell count did not change significantly as the staining time continued (i.e., 60 to 600 seconds), demonstrating that the dye stains cells quickly and readily. Furthermore, propidium iodide achieved a staining efficiency of approximately 90 cells per treatment period (30 to 600 seconds), making it a less preferred dye and subject to subsequent optimization. Compared to these two dyes, Sybr Green successfully stained only a suboptimal number of cells (approximately 75). Similarly, thiazole orange also stained approximately 85 cells within a short time period (30 seconds), but the observed cell count increased over time, demonstrating the slow staining rate of thiazole orange. Therefore, Sybr Green and thiazole orange were no longer considered primary dyes for the development of the compound formulation.

[0058] Furthermore, to examine the stability of fluorescent dye staining, we exposed the stained samples to excitation light for 10 minutes and observed changes in fluorescence intensity. As shown in Figure 2, cells stained with Sybr Gold and propidium iodide showed no significant change in fluorescence intensity after prolonged exposure. However, cells stained with Sybr Green and thiazole orange showed no weak fluorescence after approximately 5 minutes of exposure, demonstrating their susceptibility to fluorescence quenching.

[0059] Therefore, we selected Sybr Gold and propidium iodide as the optimal reagents for the fluorescent dye compound of the kit.

[0060] Example 2: Optimization of the concentration of the main fluorescent dye

[0061] In order to determine the optimal dye concentration, somatic cells in milk were stained with different concentrations of fluorescent dyes, and the number of stained cells was then examined to determine the optimal dye concentration.

[0062] The specific operation is as follows: 10000× Sybr Gold stock solution was diluted with 10mM pH 7.4 PBS solution (containing 1% (v / v) Triton X-100, 10% (v / v) glycerol, 4mg / mL EDTA-disodium, 0.3mg / mL potassium sorbate) to prepare 2, 8, 12, 16, 20 and 40× solutions respectively. During the test, 50μL of the above dilutions were mixed with 50μL of milk sample (231 standard samples / μL sample), shaken for 10s, and 8μL of the mixture was dropped onto a card for microscopic observation. The test was repeated three times, and five photos were taken each time for counting.

[0063] A 10 mg / mL propidium iodide stock solution was diluted with a 10 mM pH 7.4 PBS solution (containing 1% (v / v) Triton X-100, 10% (v / v) glycerol, 4 mg / mL disodium EDTA, and 0.3 mg / mL potassium sorbate) to prepare solutions of 20, 100, 200, 400, and 800 μg / mL, respectively. During the test, 50 μL of the above dilutions were mixed with 50 μL of milk sample (231 standard samples / μL sample), shaken for 10 seconds, and 8 μL of the mixture was dropped onto a card for microscopic observation. This was repeated three times, and five photos were taken each time for counting.

[0064] The results are shown in Figure 3. The observed cell number peaked at a Sybr Gold concentration of 12×. Therefore, Sybr Gold 12× is the optimal staining concentration. Furthermore, the observed cell number peaked at a propidium iodide concentration of 200 μg / mL, suggesting that 200 μg / mL is the optimal propidium iodide staining concentration.

[0065] Example 3: Optimization of surfactant concentration

[0066] The optimal concentration of surfactant was determined by adding different concentrations of surfactant to the fluorescent dye system to stain somatic cells in milk and then examining the number of cells observed after staining.

[0067] The specific operation is as follows: use 10mM pH 7.4 PBS solution (containing 12×Sybr Gold, 10% (v / v) glycerol, 4mg / mL EDTA-disodium, 0.3mg / mL potassium sorbate) to prepare mixed solutions with Triton X-100 content of 0%, 0.1%, 0.5%, 1%, 3%, 5%, and 10% (v / v). During the test, 50μL of the above mixed solution and 50μL of milk sample (878 standard substances / μL) were mixed to a total volume of 100μL. After shaking for 10s, 8μL of the mixed solution was dropped onto a card for microscopic observation. The test was repeated three times, and five photos were taken each time for counting.

[0068] Mixed solutions with Triton X-100 contents of 0%, 0.1%, 0.5%, 1%, 3%, 5%, and 10% (v / v) were prepared using 10 mM PBS solution (containing 200 μg / mL propidium iodide, 10% (v / v) glycerol, 4 mg / mL disodium EDTA, and 0.3 mg / mL potassium sorbate). During the test, 50 μL of the above mixed solution was mixed with 50 μL of milk sample (878 standard cells / μL) to a total volume of 100 μL. The mixture was shaken for 10 s, and then 8 μL of the mixed solution was dropped onto a card for microscopic observation. The test was repeated three times, and five photos were taken each time for counting.

[0069] The results, as shown in Figure 4, show that when the Sybr Gold dye system contains a 5% (v / v) concentration of Triton X-100, the observed cell count reaches a peak. Therefore, adding 5% (v / v) Triton X-100 to the dye formulation is the preferred surfactant concentration. Similarly, adding 1% (v / v) Triton X-100 to propidium iodide can cause the cell count to reach a peak. Notably, at the optimal surfactant concentration (i.e., adding 1% (v / v) Triton X-100), propidium iodide's ability to stain cells increases dramatically, with the observed cell count being approximately 150, comparable to the number of cells stained by Sybr Gold, demonstrating similarly excellent cell staining ability.

[0070] Example 4: Optimization of pH value of dye system

[0071] In this experiment, the pH value was adjusted and different dyes were used to stain the somatic cells in milk under different pH conditions to optimize the optimal pH value for staining with different dyes.

[0072] The specific operation is as follows: 10000× Sybr Gold stock solution was prepared into 12× solution using PBS solution (10mM) with pH 6.0, 6.5, 7.0, 7.4 and 8.0 (containing 5% (v / v) Triton X-100). During the test, 50μL of the above dilution was mixed with 50μL of milk sample (465 standard units / μL), shaken for 10s, and 8μL of the mixture was dropped onto a card for microscopic observation. The test was repeated three times, and five photos were taken each time for counting.

[0073] A 10 mg / mL propidium iodide stock solution was prepared into 200 μg / mL solutions using PBS solutions (10 mM) at pH 6.0, 6.5, 7.0, 7.4, and 8.0 (containing 1% (v / v) Triton X-100). During the test, 50 μL of the above dilutions were mixed with 50 μL of milk sample (465 standard units / μL), shaken for 10 seconds, and 8 μL of the mixture was dropped onto a card for microscopic observation. This was repeated three times, and five photos were taken each time for counting.

[0074] The results are shown in Figure 5. Sybr Gold staining of somatic cells reaches its maximum value when the pH is ≥ 7.4, thus making pH 7.4 the optimal value for this dye. Observing the propidium iodide dye system, the somatic cell staining ability was not significantly affected by pH values ​​ranging from 6.0 to 8.0. Therefore, a pH range of 6.0 to 8.0 was acceptable. Ultimately, pH 7.4, the most commonly used cell buffer system, was selected as the optimal pH for both dye systems.

[0075] Example 5: Optimization of the temperature effect of the dye system

[0076] In order to investigate the effect of temperature on the dye system, the dyeing performance of the dye system at different temperatures on somatic cells in milk was compared.

[0077] The specific operation is as follows: 10000× Sybr Gold mother solution is used to prepare a 12× solution (aqueous solution containing 5% (v / v) Triton X-100), and the 12× Sybr Gold solution and 2mL milk sample (standard 465 / μL) are incubated in a water bath at 25, 30, 35, 40, 50, and 60°C for 5 minutes respectively. During the test, 50μL of the above dilution solution is mixed with 50μL of milk sample, shaken for 10 seconds, and 8μL of the mixture is dropped onto a card for observation under a microscope. Repeat three times, and take five photos each time for counting.

[0078] Prepare a 200 μg / mL solution (aqueous solution containing 1% (v / v) Triton X-100) from a 10 mg / mL propidium iodide stock solution. Incubate the 200 μg / mL propidium iodide solution and 2 mL of milk sample (standard 465 / μL) in a water bath at 25, 30, 35, 40, 50, and 60°C for 5 minutes. During the test, take 50 μL of the above dilution and mix it with 50 μL of milk sample, shake it for 10 seconds, and take 8 μL of the mixture and drop it on a card for observation under a microscope. Repeat three times, taking five photos and counting each time.

[0079] The results are shown in Figure 6. As the temperature increases, no significant change in the number of cells stained with the two dyes is observed, demonstrating that temperature has no significant effect on the staining performance of Sybr Gold and propidium iodide dyes (p>0.05). Therefore, room temperature can be used for staining.

[0080] Example 6: Optimization of EDTA-disodium salt and glycerol concentrations

[0081] In order to avoid cell aggregation and ensure that cells can quickly settle and no longer move in the observation field, so as to achieve good observation and counting effects, the experiment investigated the effect of EDTA-disodium salt on cell aggregation rate and the effect of glycerol on cell sedimentation time.

[0082] The specific operation is as follows: freshly collected milk mastitis samples (mastitis samples or normal milk that has been aged for a long time have a relatively high aggregation rate, while fresh normal milk samples have a very low aggregation rate) are mixed with 0, 1, 2, 4, 6, and 8 mg / mL EDTA-disodium salt solution (prepared in 10 mM pH 7.4 PBS solution) in a 1:1 (v / v) ratio, shaken for 30 seconds, and 10 μL is dropped on a cell counting plate to observe and estimate the cell aggregation. The aggregation rate is calculated as the ratio of the agglomerated cells to the total number of cells in the field of view.

[0083] A milk somatic cell sample with relatively good dispersion was taken and mixed with 0%, 5%, 10%, 16%, and 24% (v / v) glycerol solution (prepared in 10 mM PBS solution, pH 7.4) in a 1:1 ratio (v / v), respectively. The mixture was shaken for 30 seconds and observed under a microscope. The time from the moment the cells were placed on the sample until they stopped moving was defined as the somatic cell sedimentation time.

[0084] As shown in Figure 7, as EDTA-disodium salt concentration increases from 0mg / mL to 4mg / mL, the milk somatic cell aggregation rate continues to decline, proving that EDTA-disodium salt has a certain anti-cell aggregation effect, and after its concentration reaches 4mg / mL, the milk somatic cell aggregation rate change is no longer obvious, therefore, the preferred EDTA-disodium salt of 4mg / mL is the most preferred solution for compound dye. Moreover, when glycerol concentration increases to 10% (v / v), milk somatic cells can be rapidly settled in 10s, and thereafter change is no longer significant, so 10% glycerol concentration is the most preferred solution for compound dye.

[0085] Example 7: Effect of polyamidoamine dendrimer (PAMAM)

[0086] As a typical nanomaterial and excellent nanoscale unimolecular surfactant, 1 nM polyamidoamine dendrimer (PAMAM) (ethylenediamine core, 4.0 generation solution, 10 wt %; purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 163442-67-9, MDL No.: MFCD00197936, specification or purity: 10 wt % in methanol) was added to the above-mentioned composite dye and observed under a microscope.

[0087] The results, as shown in Figure 8, show that the cell dispersion is significantly enhanced, and the cell nuclei are retained after staining. Furthermore, after flowing through the subsequent microfluidic pipeline device, the particles are less likely to settle within the microfluidic pipeline, providing excellent anti-fouling effect within the pipeline.

[0088] Example 8: Effect of different fat contents on dyeing effect

[0089] The fat content of fresh whole milk is generally between 3% and 3.4%, and the highest fat content in milk currently available in China is 4.6%. In order to investigate the effect of fat content on dyeing, milk samples with different fat contents were prepared using cream and skim milk powder as a matrix.

[0090] The specific operation is as follows: cream with a fat content of 30% purchased from a supermarket is diluted with a 3.8% skim milk powder matrix (aqueous solution) to 0%, 2.0%, 3.5%, 4.0%, 6.0% and 8.0% milk powder samples, and equal amounts of high-concentration milk mastitis samples with appropriate counting are added in sequence. When testing the performance of Sybr Gold compound dye, 50 μL of the above milk sample is mixed with 50 μL of 12×Sybr Gold compound dye solution to a total volume of 100 μL, shaken for 10 seconds, and 8 μL is dropped onto a card for observation and counting under a fluorescence microscope; when testing the performance of propidium iodide compound dye, 50 μL of the above milk sample is mixed with 50 μL of 200 μg / mL propidium iodide compound dye solution to a total volume of 100 μL, shaken for 10 seconds, and 8 μL is dropped onto a card for observation and counting under a fluorescence microscope; the commercial Sofia Green dye was used as a comparison. During the test, 50 μL of the above milk sample was mixed with 50 μL of 100 μg / mL Sofia Green dye solution to a total volume of 100 μL. The mixture was shaken for 10 s, and 8 μL was dropped onto a card and counted using a fluorescence microscope.

[0091] The results are shown in Figure 9. Statistical analysis revealed no significant difference in somatic cell counts after Sybr Gold and propidium iodide staining (p>0.05). Therefore, varying fat concentrations had no significant effect on the staining efficacy of Sybr Gold and propidium iodide, as evidenced by a lack of significant changes in cell count. Compared to the commercially available Sofia Green dye, the number of cells stained with Sofia Green decreased significantly with increasing fat concentration (p<0.05), demonstrating that the developed dye system can be used for somatic cell staining in high-fat milk.

[0092] Example 9: Effect of different sugar contents (lactose, glucose) on dyeing effect

[0093] (1) Effect of different lactose contents on dyeing effect.

[0094] The lactose content in fresh milk is generally between 4.5% and 5.0%. In order to investigate the effect of lactose content on the dyeing effect, lactose was added to 3.8% skimmed milk powder matrix (aqueous solution) to prepare milk samples with lactose contents (mass percentage) of 0.0%, 2.0%, 4.0%, 6.0% and 8.0%, respectively. Equal amounts of high-concentration milk mastitis samples with appropriate counting were added in sequence. When testing the performance of Sybr Gold compound dye, 50 μL of the above milk sample was mixed with 50 μL of 12×Sybr Gold compound dye solution to a total volume of 100 μL, shaken for 10 seconds, and 8 μL was dropped onto a card for observation and counting under a fluorescence microscope. When testing the performance of propidium iodide compound dye, 50 μL of the above milk sample was mixed with 50 μL of 200 μg / mL propidium iodide compound dye solution to a total volume of 100 μL, shaken for 10 seconds, and 8 μL was dropped onto a card for observation and counting under a fluorescence microscope. The commercial Sofia Green dye was used as a comparison. During the test, 50 μL of the above milk sample was mixed with 50 μL of 100 μg / mL Sofia Green dye solution to a total volume of 100 μL. The mixture was shaken for 10 s, and 8 μL was dropped onto a card and counted using a fluorescence microscope.

[0095] The results are shown in Figure 10. There was no significant difference in the number of somatic cells after staining with Sybr Gold, propidium iodide, and commercial Sofia Green (p>0.05). Therefore, different lactose contents had no significant effect on the staining effects of Sybr Gold, propidium iodide, and commercial Sofia Green dyes, as reflected in the lack of significant changes in cell number. This proves that the dye systems developed this time can be used for somatic cell staining in milk with high lactose content.

[0096] (2) The effect of different glucose contents on dyeing effect.

[0097] The glucose content in normal human serum is generally between 3.9 and 6.0 mmol / L. In order to simulate the influence of complex matrix serum environment on dye staining effect, glucose was added to 60 mg / mL bovine serum albumin (BSA) solution to prepare samples with glucose content of 0, 3, 6, 9 and 12 mmol / L, respectively. Equal amounts of high-concentration milk mastitis samples with appropriate counting were added in sequence. When testing the performance of Sybr Gold compound dye, 50 μL of the above milk sample was mixed with 50 μL of 12× Sybr Gold compound dye solution to a total volume of 100 μL, shaken for 10 seconds, and 8 μL was dropped onto a card for observation and counting under a fluorescence microscope. When testing the performance of propidium iodide compound dye, 50 μL of the above milk sample was mixed with 50 μL of 200 μg / mL propidium iodide compound dye solution to a total volume of 100 μL, shaken for 10 seconds, and 8 μL was dropped onto a card for observation and counting under a fluorescence microscope. The commercial Sofia Green dye was used as a comparison. During the test, 50 μL of the above milk sample was mixed with 50 μL of 100 μg / mL Sofia Green dye solution to a total volume of 100 μL. The mixture was shaken for 10 s, and 8 μL was dropped onto a card and counted using a fluorescence microscope.

[0098] The results are shown in Figure 11. There was no significant difference in the number of somatic cells after staining with Sybr Gold and propidium iodide at different glucose levels (p>0.05). However, the number of cells stained with the commercial Sofia Green dye decreased significantly with increasing glucose concentration (see glucose concentration of 6 mmol / L, p<0.05), demonstrating that the staining system developed this time can be used for staining stromal cells with high glucose content.

[0099] Example 10: Effect of different protein contents on dyeing effect

[0100] The protein content in fresh milk is generally between 2.5% and 4.0%. In order to investigate the effect of protein content on the dye staining effect, bovine serum albumin (BSA) was added to water to prepare matrix solutions with protein contents of 0.0%, 1.5%, 3.0%, 4.5%, 6.0% and 7.5%, respectively. Equal amounts of high-concentration milk mastitis samples with appropriate counting were added in sequence. When testing the performance of Sybr Gold compound dye, 50 μL of the above milk sample was mixed with 50 μL of 12× Sybr Gold compound dye solution to a total volume of 100 μL, shaken for 10 s, and 8 μL was dropped onto a card for observation and counting under a fluorescence microscope. When testing the performance of propidium iodide compound dye, 50 μL of the above milk sample was mixed with 50 μL of 200 μg / mL propidium iodide compound dye solution to a total volume of 100 μL, shaken for 10 s, and 8 μL was dropped onto a card for observation and counting under a fluorescence microscope. Commercial Sofia Green dye was used as a comparison. When testing, 50 μL of the above milk sample was mixed with 50 μL of 100 μg / mL The Sofia Green dye solution was mixed to a total volume of 100 μL, shaken for 10 s, and 8 μL was dropped onto the card for observation and counting under a fluorescence microscope.

[0101] The results are shown in Figure 12. Different protein contents had no significant effect on the staining effects of Sybr Gold, propidium iodide, and commercial SofiaGreen (p>0.05), which was reflected in the lack of significant changes in cell number. This proves that the two dye systems developed this time can be used for somatic cell staining in high-protein milk.

[0102] Example 11: Validation of Methodology

[0103] In order to verify the dyeing performance of the developed compound dye, a spiked recovery experiment was carried out.

[0104] First, standard samples of milk somatic cells at three concentrations (130, 465 and 1085 cells / μL) were prepared, that is, the standard somatic cells were added to skim milk powder.

[0105] Then, 50 μL of the above milk sample and 50 μL of the developed compound dye (Sybr Gold and propidium iodide) solution were mixed and shaken for 10 s. 10 μL was dropped onto a cell counting plate and counted using a fluorescence microscope. The recovery rate was calculated. The results are shown in Table 1. The recovery rates of the Sybr Gold group were between 96% and 109%, and the recovery rates of the propidium iodide group were between 94% and 108%.

[0106] Table 1. Spiked recovery of two milk somatic cell dyes

[0107] Therefore, the recovery rates of the two groups were good, and the relative standard deviations were less than 9%, which proved that the two somatic cell dye compound developed had excellent staining performance.

Claims

1. A compound dye for fluorescent staining of cells in complex matrices, characterized in that: The formula includes the following based on a total volume of 100 mL: Buffer system with pH of 6.0 to 8.0; Fluorescent dye, wherein the fluorescent dye is Sybr Gold with a final concentration of 2× to 40× or propidium iodide 2 to 80 mg; Triton X-100 0.1~10mL; Glycerol 0-24 mL; EDTA-disodium 0~800mg.

2. The compound dye for fluorescent staining of cells in a complex matrix according to claim 1, characterized in that Also included are polyamidoamine dendrimers at a final concentration of 0 to 1 nM.

3. The compound dye for fluorescent staining of cells in a complex matrix according to claim 1, characterized in that The buffer system is a phosphate buffer system, a Tris-HCl buffer system or a HEPES buffer system; And / or, the buffer system comprises sodium chloride for maintaining cell osmotic pressure.

4. The compound dye for fluorescent staining of cells in a complex matrix according to claim 1, characterized in that The fluorescent dye is Sybr Gold 8× to 16× or propidium iodide 10 to 40 mg.

5. The compound dye for fluorescent staining of cells in a complex matrix according to claim 4, characterized in that The fluorescent dye is Sybr Gold 12× or propidium iodide 20 mg.

6. The compound dye for fluorescent staining of cells in a complex matrix according to claim 1, characterized in that In every 100 mL of total volume, the volume of Triton X-100 is 1 to 5 mL; glycerol is 5 to 16 mL; and EDTA-disodium is 200 to 600 mg.

7. The compound dye for fluorescent staining of cells in a complex matrix according to claim 6, characterized in that For every 100 mL of total volume, 10 mL of glycerol; 400 mg of EDTA-disodium; When the fluorescent dye is Sybr Gold, the volume of Triton X-100 is 5 mL; when the fluorescent dye is propidium iodide, the volume of Triton X-100 is 1 mL.

8. The compound dye for fluorescent staining of cells in a complex matrix according to claim 1, characterized in that Also includes preservatives.

9. A kit for fluorescent staining of cells in complex matrices, characterized in that: The invention comprises the compound dye according to any one of claims 1 to 8.

10. A cell fluorescence staining method, characterized in that: The following steps are involved: After uniformly mixing the compound dye according to any one of claims 1 to 8 with a cell sample to be tested, the sample is taken and observed under a fluorescence microscope.

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