Compound dye for fluorescent staining of cells in complex matrix

Abstract

The invention discloses a compound dye for fluorescent staining of cells in a complex matrix. The formula of the compound dye comprises a buffer system with the pH value of 6.0-8.0, a dye solution with the pH value of 6.0-8.0 and a dye solution with the total volume of 100 mL, the fluorescent dye is Sybr Gold 2 * (40 *) or 2 to 80 mg of propidium iodide; and 0.1 to 10 mL of triton X-100, and 0.1 to 10 mL of a 0 to 24 mL of glycerol; 0 to 800 mg of EDTA (Ethylene Diamine Tetraacetic Acid)-disodium; the invention relates to a polyamide-amine dendritic polymer. According to the two preferable kit compound fluorescent dye formulas, efficient dyeing of cells in a complex matrix and accurate counting of the number of the cells can be achieved, and the dye has excellent fluorescence developing capacity. Compared with other dyes, the compound dye has the advantages of high dyeing rate (dyeing can be carried out within 30 seconds), stable dyeing, long-time illumination and difficulty in fluorescence quenching.

Description

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 science formed by the combination of cytology and chemistry. It is based on cell morphology and comprehensively applies the principles of chemical reactions to conduct qualitative, localized, and semi-quantitative analysis of various chemical components in cells (such as enzymes, esters, sugars, metal elements, proteins, nucleic acids, etc.). Among them, cell fluorescence staining technology is extremely important for detecting the number of cells in complex matrices.

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

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

[0005] Therefore, for cell counting 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 fluorescent signals after fluorescent staining, while non-target particles (proteins, fat particles, etc.) have no signals or the signals are almost negligible. Therefore, weakening non-target signals can reduce their impact on the test results, making the cell counting results more accurate.

[0006] However, existing cell fluorescent dye systems (such as ethidium bromide, thiazole orange, etc.) often have disadvantages such as low staining intensity, low staining efficiency (staining rate <70% within 10 min), slow staining rate (usually staining time >30 min), unstable fluorescent dyes (prone to fluorescence quenching), and high toxicity, which can cause 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 staining time required 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 in a 1:1 (v:v) ratio or even a larger multiple, which complicates the operation process and increases the overall detection time.

[0008] In addition, in order to accurately count cells in complex matrices, lengthy separation methods (such as membrane separation, gradient centrifugation, chromatographic column separation, electrophoresis separation, etc.) are usually required to remove interfering matrices, followed by staining and counting of cells, 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 milk samples, thereby causing somatic cell aggregation. The number of somatic cells in milk is an important indicator of milk safety and quality. A high number of somatic cells 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] In order to solve the above problems, it is necessary to develop a fluorescent staining compound and kit with good and rapid staining effect, so as to achieve rapid and stable staining of cells in complex systems, so as to meet 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 a complex matrix, aiming to achieve rapid and stable staining of cells to meet the purpose of accurate counting of cells in a complex matrix.

[0011] The present invention first provides a compound dye for fluorescent staining of cells in a complex matrix, the formula of which includes, based on a total volume of 100 mL: Buffer system with pH of 6.0~8.0; 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 0.1~10 mL; Glycerol 0~24 mL; EDTA-disodium 0~800 mg.

[0012] The fluorescent dye Sybr Gold was purchased from Beijing Langbolide Trading Co., Ltd., item number CG002-500 μL, 500 μL / bottle, the mother solution concentration was 10000×, diluted 10000 times to 1×, diluted 5000 times to 2×, and diluted 250 times to 40×.

[0013] Preferably, the composite dye further comprises a polyamide-amine dendrimer at a final concentration of 0 to 1 nM. Polyamide-amine dendrimer (PAMAM) currently has products of generation 0 to 10 (G0-G10.0). Any generation of products can be used in this application, preferably G4.0 generation products. Preferably, the final concentration of the polyamide-amine dendrimer is 1 nM.

[0014] Preferably, the buffer system is a phosphate buffer system, a Tris-HCl buffer system or a HEPES buffer system; And / or, the buffer system contains sodium chloride for maintaining the cell osmotic pressure. The general 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.

[0015] "And / Or" means that you can choose "and" or "or". Choosing "and" means both conditions need to be met, while choosing "or" means choosing one of them.

[0016] 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, with 22.8 mg of sodium dihydrogen phosphate, 115 mg of sodium dihydrogen phosphate, and 0.9 g of sodium chloride.

[0017] Preferably, the fluorescent dye is Sybr Gold 8×~16× or propidium iodide 10~40 mg.

[0018] Further preferably, the fluorescent dye is Sybr Gold 12× or propidium iodide 20 mg.

[0019] Preferably, per 100 mL total volume, the volume of Triton X-100 is 1-5 mL; glycerol is 5-16 mL; and EDTA-disodium is 200-600 mg.

[0020] Further preferably, in every 100 mL 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.

[0021] In order to prevent the product from deteriorating and to preserve it for a long time, preferably, the compound dye for fluorescent staining of cells in the complex matrix also includes a preservative. The preservative can use the type and amount of existing commonly used preservatives, such as 30 mg of potassium sorbate can be added to 100 mL of total volume.

[0022] The present invention further provides a kit for fluorescent staining of cells in complex matrices, comprising the compound dye.

[0023] The present invention also provides a cell fluorescence staining method, comprising the following steps: after mixing the compound dye with the cell sample to be tested, sampling and placing under a fluorescence microscope for observation. Generally, the compound dye and the cell sample to be tested can be mixed in a volume ratio of 1:1. During mixing, the mixture can be oscillated by a vortex oscillator so as to be quickly and fully mixed. The vortex oscillator can be used for 10 s of oscillation mixing time.

[0024] The cell sample to be tested is preferably milk or blood. The matrix components in milk or blood samples are complex, but the compound dye of the present invention is still applicable.

[0025] The components of the composite dye for fluorescent staining of cells in complex matrices of the present invention have the following functions: Sybr Gold or ethidium iodide is the main dye component and plays a major role. Sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride are used as pH buffers to buffer the solution and maintain the osmotic pressure of the cells. Triton X-100 is used as a surfactant to enhance the permeability and staining effect. Glycerol allows the stained cells to settle quickly during observation, achieving the purpose of good observation and counting in a short time. Disodium ethylenediaminetetraacetic acid (EDTA-disodium) is used to reduce the aggregation rate between cells to achieve accurate counting. Polyamidoamine dendrimer (PAMAM) is used as a nano-surfactant to achieve the purpose of cell wrapping by using its surface electrical properties to enhance cell dispersion, while keeping the stained cell nuclei from dispersing. Moreover, when flowing in subsequent microfluidic pipelines and other flow paths, it can play a role in surface antifouling and enhance the reusability of the pipeline. Potassium sorbate plays a role in corrosion protection, making the sample less likely to deteriorate.

[0026] Beneficial effects of the present invention: (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 fluorescent color development capabilities.

[0027] (2) Compared with other dyes, this composite dye has a fast dyeing rate (dyeing can be completed within 30 s) and is stable in staining. It is not prone to fluorescence quenching even under long-term illumination. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1This is a test result of the effect of main 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, and there is no significant difference; different letters represent p<0.05, and there is a significant difference. In addition, in the significance analysis, only the analysis within the same group is performed, and the analysis between different groups is not performed. The following figures are equivalent.

[0029] Figure 2 These are the effects of milk somatic cell samples stained with different dyes after continuous exposure to excitation light for 30 s, 5 min, and 10 min.

[0030] Figure 3 This is a graph showing the changes in cell number observed after staining with two dyes at different concentrations.

[0031] Figure 4 This is a graph showing the effect of surfactant concentration on cells stained with different dyes.

[0032] Figure 5 This is a graph showing the effect of pH on dye chromosome cells.

[0033] Figure 6 This is a graph showing the effect of temperature on dye chromosome cells.

[0034] Figure 7 The effects of EDTA-disodium salt on the aggregation rate of somatic cells and glycerol concentration on the sedimentation time of somatic cells are shown.

[0035] Figure 8 This is the test result of the effect of PAMAM addition on the dye, where: Figure 8 A and B in the figure represent the 12× Sybr Gold compound dye without and with polyamidoamine dendrimer, respectively; Figure 8 C and D in the figure represent the case where no polyamidoamine dendrimer was added or added to 200 µg / mL propidium iodide composite dye, respectively; Figure 8 Figures E and F in the figure respectively represent the adhesion of polyamidoamine dendrimers to the inner wall of the microfluidic channel without or with the addition of dye.

[0036] Fig. 9 This is a graph showing the test results of the effect of fat content in milk on the cell staining effect of dyes.

[0037] Fig.10 This is a graph showing the test results of the effect of lactose content in milk on dye-stained cells.

[0038] Fig.11 This is a graph showing the test results of the effect of different glucose concentrations on dye-stained cells in a serum environment simulated by bovine serum albumin.

[0039] Fig.12 This is a graph showing the effect of different protein contents on dye-stained cells. DETAILED DESCRIPTION

[0040] The optimal compound formulation of the two cell fluorescence kits is as follows: S1 compound: 120 μL of 10000× Sybr Gold (the main fluorescent dye, purchased from Beijing Langbolide 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 a 0.1 μM solution first, take 14.2 mg of PAMAM original solution (ethylenediamine core, 4.0 generation, internal concentration 10 wt%) and dissolve in 1 L of ultrapure water; the final concentration in the compound is 1 nM.) and 30 mg of potassium sorbate, dilute to 100 mL in a volumetric flask with ultrapure water, and mix thoroughly (the excitation wavelength of the fluorescent dye in this formula is 495 nm, and the emission wavelength is around 537 nm).

[0041] S2 compound: Add 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 first, and take 14.2 mg of PAMAM original solution (ethylenediamine core, 4.0 generation, internal concentration 10 wt%) in 1 L of ultrapure water; the final concentration in the compound is 1 nM.) and 30 mg of potassium sorbate, make up to 100 mL in a volumetric flask with ultrapure water, and mix thoroughly (the excitation wavelength of the fluorescent dye in this formula is about 535 nm, and the emission wavelength is about 617 nm).

[0042] The difference between the S1 compound and the S2 compound lies in the different amounts of the main fluorescent dye and surfactant used, while the other ingredients are the same, specifically: 120 μL Sybr Gold and 5 mL Triton X-100 in S1, and 20 mg propidium iodide and 1 mL Triton X-100 in S2.

[0043] Here’s how to use it: When in use, mix the prepared compound dye with the cell sample to be tested at a ratio of 1:1 (v / v), vortex on a vortex shaker for 10 s to fully mix it, drop the sample onto a standard card (or slide, cell counting plate), and observe it under a fluorescence microscope.

[0044] Example 1: Screening of major fluorescent dyes

[0045] In order to screen out the main fluorescent dyes with the best performance and the best effect (i.e., fast staining rate and large 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 counted the average number of cells that could be observed in the microscope field after staining. The specific operation was: 2×Sybr Gold, 100 µg / mL propidium iodide, 2×Sybr Green, 2 μM thiazole orange and 465 / μL milk standard samples were added to the test tube in a volume of 50 μL+50 μL and shaken for 10 s, 8 μL of the mixed sample was dropped on the standard card, and the count was observed on a fluorescent inverted microscope.

[0046] The results are as follows Figure 1 As shown in the figure, Sybr Gold dye can make the number of cells stained reach the maximum value of about 100 within 30 s. As the time continues to extend (i.e., 60-600 s), the number of cells observed does not change significantly, proving that the dye has a fast staining rate and the cells are easy to be stained. In addition, the staining efficiency of propidium iodide-stained cells is about 90 at any time (30-600 s), which can be used as a suboptimal dye and can be optimized later. Compared with these two dyes, the number of cells successfully stained by Sybr Green is not good (about 75); similarly, the number of cells stained by thiazole orange dye in a short time (30 s) is not ideal, about 85, but as time goes on, the number of cells observed increases, proving that thiazole orange has a slow staining rate for cells. Therefore, Sybr Green and thiazole orange are no longer the main dyes for the development of compound optimization.

[0047] In addition, in order to investigate the stability of fluorescent dye staining, we continuously exposed the stained samples to the presence of excitation light for 10 min and observed the changes in fluorescence intensity. Figure 2 As shown in the figure, the observation results of cells stained with Sybr Gold and propidium iodide did not change significantly after long-term exposure. The cells stained with Sybr Green and thiazole orange no longer showed weak fluorescence after about 5 minutes of exposure, proving that fluorescence quenching is prone to occur.

[0048] Therefore, we selected Sybr Gold and propidium iodide as the optimal reagents for the fluorescent dye complex of the kit.

[0049] Example 2: Optimization of the concentration of the main fluorescent dye

[0050] In order to determine the optimal dye staining 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 staining concentration.

[0051] The specific operation is as follows: 10000× Sybr Gold stock solution was diluted with 10 mM pH 7.4 PBS solution (containing 1% (v / v) Triton X-100, 10% (v / v) glycerol, 4 mg / mL EDTA-disodium, 0.3 mg / mL potassium sorbate) to prepare 2, 8, 12, 16, 20 and 40× solutions. 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 s, and 8 μL of the mixed solution was dropped on the card for observation under a microscope. The test was repeated three times, and five photos were taken each time for counting.

[0052] The 10 mg / mL propidium iodide stock solution was diluted with 10 mM pH 7.4 PBS solution (containing 1% (v / v) Triton X-100, 10% (v / v) glycerol, 4 mg / mL EDTA-disodium, 0.3 mg / mL potassium sorbate) to prepare 20, 100, 200, 400 and 800 µg / mL solutions. 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 s, and 8 µL of the mixture was dropped on the card for microscopic observation. The test was repeated three times, and five photos were taken each time for counting.

[0053] The results are as follows Figure 3 As shown in the figure, when the Sybr Gold concentration was 12×, the observed cell number reached a peak. Therefore, Sybr Gold 12× is the optimal staining concentration. In addition, when the propidium iodide concentration was 200 µg / mL, the observed cell number reached a peak, and it is believed that the optimal staining concentration of propidium iodide is 200 µg / mL.

[0054] Example 3: Optimization of surfactant concentration

[0055] The optimal concentration of the surfactant was determined by adding different concentrations of surfactant to the fluorescent dye system to stain the somatic cells in milk and then examining the number of cells observed after staining.

[0056] The specific operation is as follows: 10 mM pH 7.4 PBS solution (containing 12× Sybr Gold, 10% (v / v) glycerol, 4 mg / mL EDTA-disodium, 0.3 mg / mL potassium sorbate) was used 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, 50 µL of the above mixed solution was mixed with 50 µL of milk sample (standard 878 / µL) to a total volume of 100 µL. After shaking for 10 s, 8 µL of the mixed solution was dropped onto the card for observation under a microscope. The test was repeated three times in parallel, and five photos were taken each time for counting.

[0057] 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 (pH 7.4) (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 samples / 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 observation under a microscope. The test was repeated three times, and five photos were taken each time for counting.

[0058] The results are as follows Figure 4 As shown in the figure, when the Sybr Gold dye system contains 5% (v / v) Triton X-100, the observed cell number can reach a peak. Therefore, 5% (v / v) Triton X-100 is added to the dye formula as the preferred surfactant concentration. Similarly, the addition of 1% (v / v) Triton X-100 to propidium iodide can make the cell number reach a peak. It is worth noting that at the optimal surfactant concentration (i.e., the addition of 1% (v / v) Triton X-100), the cell staining ability of propidium iodide increased dramatically, and the number of cells observed was about 150, which is comparable to the number of cells stained by Sybr Gold, and also shows a relatively excellent cell staining ability.

[0059] Example 4: Optimization of pH value of dye system

[0060] In this experiment, the pH value was adjusted and various 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.

[0061] The specific operation is as follows: 10000× Sybr Gold stock solution was used to prepare 12× solution using PBS solution (10 mM) with pH 6.0, 6.5, 7.0, 7.4 and 8.0 (containing 5% (v / v) Triton X-100). During the test, 50 µL of the above dilution was mixed with 50 µL of milk sample (standard 465 / µL), shaken for 10 s, and 8 µL of the mixture was dropped onto the card for observation under a microscope. The test was repeated three times, and five photos were taken each time for counting.

[0062] 10 mg / mL propidium iodide stock solution was prepared into 200 µg / mL solution using PBS solution (10 mM) (containing 1% (v / v) Triton X-100) at pH 6.0, 6.5, 7.0, 7.4 and 8.0, respectively. During the test, 50 µL of the above dilution was mixed with 50 µL of milk sample (standard 465 / µL), shaken for 10 s, and 8 µL of the mixture was dropped onto the card for observation under a microscope. The test was repeated three times, and five photos were taken each time for counting.

[0063] The results are as follows Figure 5 As shown in the figure, when pH ≥ 7.4, the number of somatic cells stained by Sybr Gold reaches the maximum value, so pH 7.4 is the optimal value for this dye. Observing the propidium iodide dye system, its pH value from 6.0 to 8.0 has no significant effect on the staining ability of somatic cells, so pH 6.0~8.0 is acceptable. Finally, the most commonly used cell buffer system pH 7.4 was selected as the optimal pH value for the two dye systems.

[0064] Example 5: Optimization of the temperature effect on the dye system

[0065] In order to investigate the effect of temperature on the dye system, the dyeing performance of the dyeing system at different temperatures on somatic cells in milk was compared.

[0066] The specific operation is as follows: 10000× Sybr Gold stock solution is used to prepare 12× solution (aqueous solution containing 5% (v / v) Triton X-100), and 12× Sybr Gold solution and 2 mL milk sample (standard 465 / µL) are incubated in a water bath at 25, 30, 35, 40, 50, and 60 ℃ for 5 min respectively. During the test, 50 µL of the above dilution is mixed with 50 µL of milk sample, shaken for 10 s, and 8 µL of the mixture is dropped on the card for observation under a microscope. The test is repeated three times, and five photos are taken each time for counting.

[0067] Use 10 mg / mL propidium iodide stock solution to prepare 200 µg / mL solution (aqueous solution containing 1% (v / v) Triton X-100), and incubate 200 µg / mL propidium iodide solution and 2 mL milk sample (standard 465 / µL) in a water bath at 25, 30, 35, 40, 50, and 60 °C for 5 min. During the test, take 50 µL of the above dilution and mix it with 50 µL milk sample, shake it for 10 s, take 8 µL of the mixture and drop it on the card for observation under a microscope, repeat three times, take five photos and count each time.

[0068] The results are as follows Figure 6 As shown in the figure, with the increase of temperature, no significant change in the number of cells was observed after staining with the two dyes, which proves 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.

[0069] Example 6: Optimization of EDTA-disodium salt and glycerol concentrations

[0070] In order to avoid cell aggregation and enable cells to quickly settle in the observation field and no longer move, so as to achieve good observation and counting effects, the effect of EDTA-disodium salt on cell aggregation rate and the effect of glycerol on cell sedimentation time were investigated in the experiment.

[0071] 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 s, and 10 µL is dropped on a cell counting plate to observe and estimate the aggregation of cells. The aggregation rate is calculated as the ratio of aggregated cells to the total number of cells in the field of view.

[0072] Take milk somatic cell samples with relatively good dispersion, mix them with 0%, 5%, 10%, 16%, and 24% (v / v) glycerol solution (prepared with 10 mM PBS solution, pH 7.4) in a ratio of 1:1 (v / v), shake for 30 s, and observe under a microscope. The time from the time the cells are just placed on the plate to the time the cells stop moving is the somatic cell sedimentation time.

[0073] The results are as follows Figure 7As shown in the figure, as the concentration of EDTA-disodium salt increases from 0 mg / mL to 4 mg / mL, the aggregation rate of milk somatic cells continues to decrease, proving that EDTA-disodium salt has a certain anti-cell aggregation effect, and after its concentration reaches 4 mg / mL, the aggregation rate of milk somatic cells no longer changes significantly, so 4 mg / mL EDTA-disodium salt is the most preferred solution for compound dyes. Furthermore, when the glycerol concentration increases to 10% (v / v), milk somatic cells can quickly settle within 10 s, and thereafter the change is no longer significant, so a 10% glycerol concentration is the most preferred solution for compound dyes.

[0074] Example 7: Effect of polyamidoamine dendrimer (PAMAM)

[0075] As a typical nanomaterial and excellent nanoscale single-molecule 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: 10wt% in methanol) was added to the above-mentioned composite dye and then observed under a microscope.

[0076] The results are as follows Figure 8 As shown, the dispersion of cells is significantly enhanced, and the cell nuclei can be kept intact after staining. And after flowing through the microfluidic pipeline equipment, it is not easy to settle in the microfluidic pipeline, which can play a good anti-fouling effect in the pipeline.

[0077] Example 8: Effect of different fat contents on dyeing effect

[0078] The fat content of fresh whole milk is generally between 3% and 3.4%, and the highest fat content of milk currently sold 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 skimmed milk powder as a matrix.

[0079] The specific operation is as follows: cream with a fat content of 30% purchased from a supermarket is diluted with a 3.8% skimmed milk powder matrix (aqueous solution) into 0%, 2.0%, 3.5%, 4.0%, 6.0% and 8.0% milk powder samples, and an equal amount 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 s, and 8 μL is dropped on 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 s, and 8 μL is dropped on a card for observation and counting under a fluorescence microscope; commercial Sofia Green dye is used as a comparison. When testing, 50 μL of the above milk sample is mixed with 50 μL of 100 The 1 μg / mL 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 and counted using a fluorescence microscope.

[0080] The results are as follows Fig. 9 As shown in the figure, the results were statistically analyzed. There was no significant difference in the number of somatic cells after Sybr Gold and propidium iodide staining (p>0.05). Therefore, different fat content concentrations had no significant effect on the staining effect of Sybr Gold and propidium iodide, which was reflected in the fact that there was no significant change in the number of cells. Compared with the commercial Sofia Green dye, the number of cells stained by Sofia Green dye decreased significantly with the increase of fat concentration (p<0.05), proving that the dye system developed this time can be used for somatic cell staining in high-fat milk.

[0081] Example 9: Effect of different sugar contents (lactose, glucose) on dyeing effect

[0082] (1) Effect of different lactose contents on dyeing effect.

[0083] The lactose content in fresh milk is generally between 4.5% and 5.0%. In order to investigate the effect of lactose content on dyeing effect, lactose was added to 3.8% skim milk powder matrix (aqueous solution) to prepare milk samples with lactose content (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 s, and 8 μL was dropped on the 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 on the card for observation and counting under a fluorescence microscope. The commercial SofiaGreen dye was used as a comparison. When testing, 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 on the card for observation and counting under a fluorescence microscope. μL of 100 μg / mL 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 and counted using a fluorescence microscope.

[0084] The results are as follows Fig.10 As shown, 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 content had no significant effect on the staining effect of Sybr Gold, propidium iodide and commercial SofiaGreen dyes, which was reflected in the lack of significant change in the number of cells. This proves that the dye system developed this time can be used for somatic cell staining in milk with high lactose content.

[0085] (2) The effect of different glucose contents on dyeing effect.

[0086] The glucose content in normal human serum is generally between 3.9 and 6.0 mmol / L. In order to simulate the effect of complex matrix serum environment on dye staining, glucose was added to 60 mg / mL bovine serum albumin (BSA) solution to prepare samples with glucose contents of 0, 3, 6, 9 and 12 mmol / L, and 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× SybrGold compound dye solution to a total volume of 100 μL, shaken for 10 s, and 8 μL was dropped on the 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 on the card for observation and counting under a fluorescence microscope. The 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 SofiaGreen dye solution to a total volume of 100 μL. After shaking for 10 s, 8 μL was dropped onto the card and counted using a fluorescence microscope.

[0087] The results are as follows Fig.11 As shown, 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), but the number of cells of the commercial Sofia Green dye decreased significantly with the increase of glucose concentration (see glucose concentration of 6 mmol / L, p<0.05), proving that the staining system developed this time can be used for staining stromal cells with high glucose content.

[0088] Example 10: Effect of different protein contents on dyeing effect

[0089] 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 with 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. The mixture was shaken for 10 s, and 8 μL was dropped on the 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. The mixture was shaken for 10 s, and 8 μL was dropped on the card for observation and counting under a fluorescence microscope. The 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 Sofia Green. Green dye solution was mixed to a total volume of 100 μL, shaken for 10 s, and 8 μL was dropped onto the card and counted using a fluorescence microscope.

[0090] The results are as follows Fig.12 As shown, different protein contents had no significant effect on the staining effects of Sybr Gold, propidium iodide and commercial Sofia Green (p>0.05), which was reflected in the fact that there was no significant change in the number of cells, proving that the two dye systems developed this time can be used for somatic cell staining in high-protein milk.

[0091] Example 11: Validation of methodology

[0092] In order to verify the dyeing performance of the developed composite dye, a spike recovery experiment was carried out.

[0093] Firstly, standard samples of milk somatic cells of three concentrations (130, 465 and 1085 cells / μL) were prepared, namely, the standard somatic cells were added into skimmed milk powder.

[0094] Then, 50 μL of the above milk sample and 50 μL of the prepared compound dye (Sybr Gold and propidium iodide) solution were mixed and shaken for 10 s. 10 μL was dropped onto the cell counting plate and counted under 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%.

[0095] Table 1. Spiked recovery experiments of two milk somatic cell dyes

[0096] Therefore, the recoveries of both groups were good, and the relative standard deviations were less than 9%, demonstrating the excellent staining performance of the two developed somatic cell dye composites.

Claims

1. A composite dye for fluorescent staining of cells in a complex matrix, characterized in that: The formula includes the following based on a total volume of 100 mL: Buffer system with pH of 6.0~8.0; 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 0.1~10 mL; Glycerol 0~24 mL; EDTA-disodium 0~800 mg.

2. The composite 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 0-1 nM.

3. The composite 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 composite dye for fluorescent staining of cells in a complex matrix according to claim 1, characterized in that: The fluorescent dye is Sybr Gold 8×~16× or propidium iodide 10~40 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 composite dye for fluorescent staining of cells in a complex matrix according to claim 1, characterized in that: In every 100 mL total volume, the volume of Triton X-100 is 1~5 mL; glycerol is 5~16 mL; EDTA-disodium is 200~600 mg.

7. The composite dye for fluorescent staining of cells in a complex matrix according to claim 6, characterized in that: In every 100 mL total volume, glycerol 10 mL; EDTA-disodium 400 mg; 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 composite 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 composite 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 the cell sample to be tested, the sample is taken and observed under a fluorescence microscope.

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