A composite dye for fluorescent staining of cells in complex matrices

Abstract

The present invention discloses a compound dye for fluorescent staining of cells in complex matrices. The compound dye comprises, based on a total volume of 100 mL, a buffer system having a pH of 6.0 to 8.0; a fluorescent dye, wherein the fluorescent dye is 2× to 40× Sybr Gold or 2 to 80 mg of propidium iodide; 0.1 to 10 mL of Triton X-100; 0 to 24 mL of glycerol; 0 to 800 mg of EDTA-disodium; and a polyamide-amine dendrimer. The two preferred compound fluorescent dye formulas of the present invention can both achieve efficient staining of cells in complex matrices and accurate counting of their number, and the dye has excellent fluorescence color development ability. Compared with other dyes, the compound dye has a fast staining rate (staining can be achieved within 30 seconds), stable staining, and is not prone to fluorescence quenching under long-term illumination.

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 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 cell numbers within complex matrices.

[0003] Typically, cell counting in simple matrices (such as phosphate buffer systems) can be performed using a hemocytometer. 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.), 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, counting cells in complex matrices typically requires specific staining with fluorescent dyes, followed by counting and analysis using a fluorescence microscope. The advantage of this method is that target cells exhibit a fluorescent signal after fluorescent staining, while non-target particles (proteins, fat particles, and others) show no or negligible signal. Therefore, reducing non-target signals can reduce their impact on test results, resulting in more accurate cell counts.

[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 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 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] Furthermore, accurate cell counts in complex matrices often require lengthy separation methods (such as membrane separation, gradient centrifugation, chromatographic columns, and electrophoresis) to remove interfering matrices. Subsequent cell staining and counting analysis significantly increases the staining workload and overall processing time. This complex and time-consuming separation method makes accurate measurement of time-sensitive samples more challenging. For example, in milk somatic cell count testing, increased testing time can lead to decreased sample quality and the aggregation of somatic cells. The somatic cell count in milk is a key indicator of milk safety and quality. A high somatic cell count often indicates mastitis or other udder diseases, which can reduce milk quality and may lead to the culling of affected cows, impacting milk production and economic profitability. Therefore, timely and effective monitoring of somatic cell count changes in milk is crucial to ensuring cow health and milk quality.

[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 6.0~8.0;

[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 0~800 mg.

[0017] The fluorescent dye Sybr Gold was purchased from Beijing Langbolid Trading Co., Ltd., with the 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 dye compound further comprises a polyamidoamine dendrimer at a final concentration of 0-1 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 hydrogen phosphate, and 0.9 g of sodium chloride.

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

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

[0025] 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.

[0026] Further preferably, per 100 mL 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 enable long-term storage, 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 a total volume of 100 mL.

[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 mixture can be vortexed 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 serves as the primary dye component, playing a key role. Sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride act as pH buffers, buffering the solution and maintaining the osmotic pressure of the cells. Triton X-100 acts as a surfactant, enhancing membrane permeability and staining. Glycerol allows stained cells to settle quickly during observation, enabling rapid observation and counting. Disodium ethylenediaminetetraacetic acid (EDTA-disodium) reduces cell aggregation, ensuring accurate counting. Polyamidoamine dendrimers (PAMAMs) act as nanosurfactants, utilizing their surface electrical properties to encapsulate cells, enhancing cell dispersion and preventing the stained nuclei from dispersing. Furthermore, they act as antifouling agents during flow through subsequent microfluidic channels, enhancing their reusability. Potassium sorbate acts as a preservative, preventing sample deterioration.

[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 s) and is stable. It is not prone to fluorescence quenching even under long-term illumination. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1This figure shows the test results of the effects of major fluorescent dyes on the number of somatic cells stained in milk at different staining times. 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 within-group analysis is performed, and no between-group analysis is performed. The following figures are equivalent.

[0038] 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.

[0039] Figure 3 The figure shows the detection results of the changes in cell number observed after staining with two dyes at different concentrations.

[0040] Figure 4 This figure shows the test results of the effect of surfactant concentration on cells stained with different dyes.

[0041] Figure 5 This is a graph showing the test results of the effect of pH on dye chromosome cells.

[0042] Figure 6 This is a graph showing the test results of the effect of temperature on dye chromosome cells.

[0043] 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.

[0044] 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 and the case where polyamidoamine dendrimer was added to 200 μg / mL propidium iodide compound dye, respectively; Figure 8 Figures E and F represent the adhesion of polyamidoamine dendrimers to the inner wall of the microfluidic channel with and without the dye, respectively.

[0045] Figure 9 This figure shows the test results of the effect of fat content in milk on the cell staining effect of dyes.

[0046] Figure 10 This figure shows the test results of the effect of lactose content in milk on dye-stained cells.

[0047] Figure 11 This 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] Figure 12 This figure shows the test results of the effect 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 / vial), 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 by taking 14.2 mg of PAMAM stock solution (ethylenediamine core, generation 4.0, containing 10 wt%) and dissolving it in 1 L of ultrapure water; the final concentration of the compound is 1 nM.), and 30 mg of potassium sorbate were added to a 100 mL volumetric flask with ultrapure water and mixed thoroughly (the excitation wavelength of the fluorescent dye in this formula is 495 nm, and the emission wavelength is around 537 nm).

[0051] 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 by first adding 14.2 mg of PAMAM stock solution (ethylenediamine core, generation 4.0, containing 10 wt%) 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 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, while the other ingredients are the same: 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, mix the prepared compound dye with the cell sample to be tested at a ratio of 1:1 (v / v), shake on a vortex shaker for 10 seconds to mix thoroughly, drop the sample onto a standard card (or slide, cell counting plate), and observe 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., rapid staining and high cell count), 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 s. 8 μL of the mixture was then dropped onto a standard card and counted under an inverted fluorescence microscope.

[0057] The results are as follows Figure 1 As shown, Sybr Gold dye reaches a maximum cell count of approximately 100 within 30 seconds. As the staining time increases (i.e., 60–600 seconds), the observed cell count does not change significantly, demonstrating that the dye stains cells quickly and readily. Furthermore, propidium iodide stains cells with a staining efficiency of approximately 90 cells per treatment period (30–600 seconds), making it a less preferred dye and subject to subsequent optimization. Compared to these two dyes, Sybr Green successfully stains a suboptimal number of cells (approximately 75). Similarly, thiazole orange stains an unsatisfactory number of cells within a short treatment period (30 seconds), at approximately 85. However, the observed number of cells increases with time, demonstrating the slow staining rate of thiazole orange. Therefore, Sybr Green and thiazole orange are no longer considered primary dyes for the development of compound formulations.

[0058] In addition, in order to investigate the stability of fluorescent dye staining, we exposed the stained samples to the presence of excitation light for 10 minutes and observed the changes in fluorescence intensity. Figure 2 As shown in the figure, cells stained with Sybr Gold and propidium iodide showed no significant change in fluorescence after long-term exposure. However, cells stained with Sybr Green and thiazole orange showed no weak fluorescence after approximately 5 minutes of exposure, demonstrating that fluorescence quenching is prone to occur.

[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: 10,000× 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, and 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) and shaken for 10 s. 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.

[0063] A 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 disodium EDTA, and 0.3 mg / mL potassium sorbate) to prepare solutions of 20, 100, 200, 400, and 800 µg / mL. For testing, 50 µL of each dilution was mixed with 50 µL of milk sample (231 standard molecules / µL sample) and shaken for 10 s. 8 µL of the mixture was then dropped onto a card for microscopic observation. This was repeated three times, and five photographs were taken each time for counting.

[0064] The results are as follows Figure 3 As shown in the figure, 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: 10 mM pH 7.4 PBS solution (containing 12× Sybr Gold, 10% (v / v) glycerol, 4 mg / mL EDTA-disodium, and 0.3 mg / mL potassium sorbate) is used to prepare mixed solutions with Triton X-100 contents of 0%, 0.1%, 0.5%, 1%, 3%, 5%, and 10% (v / v). During the test, 50 μL of the above mixed solution and 50 μL of milk sample (878 standard cells / μL) are mixed to a total volume of 100 μL. The mixture is shaken for 10 s, and then 8 μL of the mixed solution is dropped onto a card for microscopic observation. The test is repeated three times, and five photos are 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 (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 testing, 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. This was repeated three times, and five photos were taken each time for counting.

[0069] The results are as follows Figure 4 As shown, when the Sybr Gold dye system contains 5% (v / v) Triton X-100, the observed cell number reaches a peak. Therefore, the addition of 5% (v / v) Triton X-100 to the dye formulation is the preferred surfactant concentration. Similarly, the addition of 1% (v / v) Triton X-100 to propidium iodide can lead to a peak in cell number. Notably, at the optimal surfactant concentration (i.e., 1% (v / v) Triton X-100), propidium iodide's cell staining ability increases dramatically, with the observed cell number approximately 150, comparable to the number of cells stained by Sybr Gold, demonstrating 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: 10,000× Sybr Gold stock solution was prepared into 12× solutions using PBS solutions (10 mM) at 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 dilutions were mixed with 50 μL of milk sample (465 standard cells / μL), shaken for 10 s, and 8 μL 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). For testing, 50 µL of each of these dilutions was mixed with 50 µL of milk sample (465 standard units / µL) and shaken for 10 s. 8 µL of the mixture was then dropped onto a card for microscopic observation. This was repeated three times, with five photographs taken each time for counting.

[0074] The results are as follows Figure 5 As shown, the number of somatic cells stained by Sybr Gold reaches a maximum 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: 10,000× Sybr Gold stock solution is used to prepare a 12× solution (aqueous solution containing 5% (v / v) Triton X-100). The 12× Sybr Gold solution and 2 mL of milk sample (standard 465 cells / µL) are incubated in a water bath at 25, 30, 35, 40, 50, and 60°C for 5 minutes respectively. During the test, 50 µL of the above dilution solution is mixed with 50 µL of milk sample and shaken for 10 seconds. 8 µL of the mixture is then dropped onto a card for microscopic observation. This is repeated three times, and five photos are taken each time for counting.

[0078] A 200 µg / mL solution (aqueous solution containing 1% (v / v) Triton X-100) was prepared from a 10 mg / mL stock solution of propidium iodide. This solution was then incubated with 2 mL of milk sample (standard: 465 counts / µL) in a water bath at 25, 30, 35, 40, 50, and 60°C for 5 min, respectively. During testing, 50 µL of the diluted solution was mixed with 50 µL of the milk sample and shaken for 10 s. 8 µL of the mixture was then dropped onto a card for microscopic observation. This was repeated three times, with five photographs taken each time for counting.

[0079] 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 when 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.

[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 PBS solution with pH 7.4) 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 aggregated cells to the total number of cells in the field of view.

[0083] Well-dispersed milk somatic cell samples were taken and mixed with 0%, 5%, 10%, 16%, and 24% (v / v) glycerol solutions (prepared in 10 mM PBS solution, pH 7.4) in a 1:1 (v / v) ratio, respectively. The samples were 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] The results are as follows Figure 7As shown in the figure, as the EDTA-disodium salt concentration increased from 0 mg / mL to 4 mg / mL, the milk somatic cell aggregation rate continued to decrease, demonstrating that EDTA-disodium salt has a certain anti-cell aggregation effect. Furthermore, after reaching a concentration of 4 mg / mL, the milk somatic cell aggregation rate no longer changed significantly, thus making 4 mg / mL of EDTA-disodium salt the optimal solution for the compound dye. Furthermore, when the glycerol concentration increased to 10% (v / v), the milk somatic cells rapidly settled within 10 s, and thereafter, no significant changes occurred, thus making 10% glycerol concentration the optimal solution for the 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 are as follows Figure 8 As shown, the dispersion of cells is significantly enhanced, and the cell nuclei can be kept intact after staining. Moreover, after flowing through the subsequent microfluidic pipeline equipment, it is not easy to settle in the microfluidic pipeline, which can play an excellent anti-fouling effect in 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 commercially available milk in China is 4.6%. To investigate the effect of fat content on dyeing, milk samples with different fat contents were prepared using a cream and skim milk powder matrix.

[0090] The specific operation is as follows: cream with a fat content of 30% purchased from the supermarket is diluted with 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 s, 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 s, and 8 μL is dropped onto 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 10 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 for observation and counting under a fluorescence microscope.

[0091] The results are as follows Figure 9 Statistical analysis of the results showed no significant difference in somatic cell counts after Sybr Gold and propidium iodide staining (p>0.05). Therefore, different fat concentrations had no significant effect on the staining effects of Sybr Gold and propidium iodide, as reflected by no significant change in cell count. Compared with 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 dye system developed in this study can be used to stain somatic cells 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%. To investigate the effect of lactose content on dye staining, lactose was added to 3.8% skim 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 sequentially. 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 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. The mixture was shaken for 10 s, and 8 μL was dropped onto a card for observation and counting under a fluorescence microscope. 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. The cells were mixed with 100 μL of 100 μg / mL Sofia Green dye solution 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.

[0095] The results are as follows Figure 10 As shown in the results, 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 SofiaGreen dyes, which was 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. 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, 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× SybrGold compound dye solution to a total volume of 100 μL. The mixture was 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. The mixture was 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 200 μg / mL propidium iodide compound dye solution to a total volume of 100 μL. 1 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. The mixture was shaken for 10 s and 8 μL was dropped onto a card for observation and counting under a fluorescence microscope.

[0098] The results are as follows Figure 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.

[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%. To investigate the effect of protein content on dye staining, 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 sequentially. 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 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. The mixture was 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 Sofia Green. The 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 as follows Figure 12 As shown in the results, 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.

[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 prepared 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 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%.

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

[0107]

[0108] Therefore, the recoveries of the two groups were good, and the relative standard deviations were less than 9%, which proved that the two somatic cell dye composites developed had excellent staining performance.

Claims

1. A cell fluorescence staining method, characterized in that: The following steps are involved: After mixing the compound dye for fluorescent staining of cells in complex matrices with the cell sample to be tested, the sample is placed under a fluorescence microscope for observation; The cell sample to be tested is milk or blood; The formula for the compound dye for fluorescent staining of cells in complex matrices 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 at a final concentration of 8× to 16× or propidium iodide at a final concentration of 10 to 40 mg; Triton X-100 1-5 mL; Glycerol 5-16 mL; EDTA-disodium 200~600 mg; A polyamidoamine dendrimer was also included at a final concentration of 1 nM.

2. The cell fluorescence staining method 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.

3. The cell fluorescence staining method according to claim 1, characterized in that: The fluorescent dye is Sybr Gold 12× or propidium iodide 20 mg.

4. The cell fluorescence staining method according to claim 1, characterized in that: For 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.

5. The cell fluorescence staining method according to claim 1, characterized in that: Also includes preservatives.

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