Single dye staining reagent and method for single step staining of multiple cells in reptile blood

Abstract

The single-dye staining reagent and method for single-step staining of multiple cells in blood of reptiles comprises a staining agent A10, a permeation buffer salt A30, a dyeing stabilizer B10 and water; the staining agent A10 is new methylene blue; the concentration of the new methylene blue is 0.3*10 ‑4 ~3.0*10 ‑4 g / ml; the permeation buffer salt A30 is used to provide a low permeation pressure relative to blood and a weak alkaline environment with a pH value of 7-8; the range of the permeation pressure is 170mosmol / kg~210mosmol / kg; the aldehyde type is chemically cross-linked with the surface protein of the cell membrane, and the staining agent A10 enters the cell at a faster rate. The low permeation environment and the weak alkaline environment with a pH value of 7-8 are ingeniously designed to be well matched with the blood cells of reptiles, and the single-step staining of the single-dye reagent for multiple different types of cells in the blood of reptiles can be completed by adding the reagent once.

Description

Technical Field

[0001] This application belongs to the field of staining technology for bright-field identification of reptile blood cells, and particularly relates to a staining reagent and method for single-step rapid staining of reptile blood in a cell suspension state, which can quickly and simultaneously stain multiple cells in reptile blood, so as to enable the identification and counting of different types of cells in reptile blood. Background Technology

[0002] Different animal species are at different stages of evolution, and the types, morphologies, and numbers of blood cells in their peripheral blood vary depending on the stage of evolution. For example, the types and morphologies of blood cells in reptiles differ from those in mammals. However, regardless of the animal, all blood cells contain red blood cells and white blood cells. Staining is necessary to identify and differentiate between red and white blood cells. The specific cell types may also vary between species, including some types of neutrophils, lymphocytes, monocytes, eosinophils, and basophils.

[0003] For staining blood cells from non-mammalian animals, blood smears are typically used. However, this process is complex, requiring the application of a suitable amount of blood to a glass slide, followed by spreading the blood with a coverslip at a 30-45 degree angle, and then air-drying. Inexperienced spreaders may cause uneven cell distribution within a single layer, affecting subsequent staining and hindering morphological assessment. Furthermore, different dyes are often used to differentiate between cell types, resulting in high material and usage costs and difficulty in maintaining the morphology of viable cells. Currently, there are no existing staining reagents for staining reptile cells in suspension for cell morphology analysis.

[0004] One of the challenges of staining cells in suspension is adjusting the ion concentration. Using the cell membrane as a semipermeable membrane, if the ion concentration in the cell suspension is hypertonic, the cells will shrink and struggle to maintain their original shape. If the ion concentration is hypotonic, the cells are easily swollen, and in severe cases, may even rupture, ultimately failing to maintain their shape and preventing the staining effect from being maintained until cell observation is complete. When the ion concentration in the suspension is isotonic, meaning external water does not enter the cell, it is relatively easy to maintain cell shape. However, in an isotonic state, the staining material also does not easily penetrate the cell nucleus for nuclear staining, requiring a longer staining time. Nevertheless, to maintain cell shape for easy observation, isotonic staining reagents are generally used in suspensions.

[0005] Definitions:

[0006] Nucleic acid substances refer to substances with genetic characteristics that exist within cells, namely DNA or RNA or their fragments.

[0007] EDTA salts refer to ethylenediaminetetraacetic acid (EDTA). EDTA binds with calcium in the blood to form coordination compounds, thereby preventing blood clotting. EDTA salts are commonly used as blood anticoagulants.

[0008] Osmotic pressure: Separated by a semipermeable membrane, with lysed water on one side and a solvent on the other, water permeates through the membrane to the solution side. The pressure applied to the solution side to prevent water movement is called osmotic pressure. Water movement stops when this pressure equals the chemical potential energy of the water passing through the membrane. Osmotic pressure is expressed as osmolality, typically in milliostobars (mIs) of solute per kilogram of solvent.

[0009] The CAS numbers of some of the substances in this application are as follows:

[0010] Sodium hydrogen phosphate 10039-32-4 Potassium dihydrogen phosphate 7778-77-0 NaCl 2647-14-5 <![CDATA[EDTA·K2]]> 2001-94-7 <![CDATA[EDTA·K3]]> 17572-97-3 <![CDATA[EDTA·Na2]]> 6381-92-6 New Asian Blue 229-516-6 water 7732-18-5 Commercial 37% formaldehyde solution 50-00-0 Commercial 50% glutaraldehyde solution 111-30-8

[0011] (CAS Registry Number, also known as CASNumber, CASRn, CAS#), or CAS Registry Number, is a unique digital identifier for a substance (compound, polymer, biological sequence, mixture, or alloy).

[0012] The water in this application is purified water. Purified water refers to water that meets the purified water standard of the Chinese Pharmacopoeia (2015 edition), and its main parameters are: when tested with an offline conductivity meter at 25°C, the conductivity is not greater than 25 μS / cm (micro-Siemens per centimeter) when the labeled volume is not greater than 10 ml.

[0013] GB / T6686-2006, the standard for dye classification, categorizes basic dyes as cationic dyes. "Basic dyes:"

[0014] Formerly known as basic dyes, these are dyes that can dissociate in aqueous solution to form cationic pigments; cationic dyes are a class of dyes that can dissociate in aqueous solution to form cationic pigments. These dyes can be used to dye substances containing anionic dyes in a weakly acidic dye bath. Summary of the Invention

[0015] To provide an efficient method for simultaneous, single-step staining of multiple cell types, this invention proposes a single-dye staining reagent and method for single-step staining of multicellular reptile blood, as well as its preparation method.

[0016] The technical solution of this application to solve the above-mentioned technical problem is a single-dye staining reagent for single-step staining of multicellular blood of reptiles, comprising staining agent A10, osmotic buffer salt A30, staining stabilizer B10, and water; staining agent A10 is neomethylene blue; the concentration of neomethylene blue is 0.3 × 10⁻⁶. -4 ~3.0×10 -4 g / ml; Osmotic buffer A30 is used to provide a low osmotic pressure relative to blood, with an osmotic pressure range of 170 mosmol / kg to 210 mosmol / kg; At the same time, osmotic buffer A30 is used to provide a weakly alkaline environment with a pH of 7-8; Staining stabilizer B10 includes aldehydes, which are used to chemically cross-link with proteins on the cell membrane surface, accelerating the rate at which staining agent A10 enters the cell.

[0017] The single-dye staining reagent for multi-cell single-step staining of reptile blood is used for staining turtle blood cells, with a pH value of 7.1 to 7.5.

[0018] The single-dye staining reagent for multi-cell single-step staining of reptile blood, comprising osmotic buffer A30, includes disodium hydrogen phosphate and potassium dihydrogen phosphate.

[0019] The single-dye staining reagent for multi-cell single-step staining of reptile blood, used for staining turtle blood cells, has a disodium hydrogen phosphate concentration of 4.3 × 10⁻⁶. -2 ~5.7×10 -2 The concentration of potassium dihydrogen phosphate is 9.5 × 10 mol / L. -3 ~2.5×10 - 2 mol / L; pH value is 7.1~7.5.

[0020] The single-dye staining reagent for multi-cell single-step staining of reptile blood, wherein the staining stabilizer B10 includes formaldehyde and glutaraldehyde.

[0021] The single-dye staining reagent for multi-cell single-step staining of reptile blood, used for turtle blood cell staining, has a formaldehyde concentration of 0.9 × 10⁻⁶. -2 mol / L~1.3×10 -2 mol / L; glutaraldehyde concentration is 3.6 × 10⁻⁶ mol / L. -3 mol / L~4.8×10 - 3 mol / L.

[0022] The single-dye staining reagent for multi-cell single-step staining of reptile blood, comprising osmotic buffer A30, further includes NaCl, with a NaCl concentration range of 4.67 × 10⁻⁶. -3 mol / L~1.98×10 -2 mol / L.

[0023] The single-dye staining reagent for multi-cell single-step staining of reptile blood contains 0.5 mL of proClean950 solution (preservative) per 500 ml of staining reagent.

[0024] The technical solution of this application to solve the above-mentioned technical problem can also be a single-step rapid staining method for reptile blood cells, using the above-mentioned single-dye staining reagent for single-step staining of multi-cell reptile blood; including the step of mixing a standard blood sample with the above-mentioned single-dye staining reagent for single-step staining of multi-cell reptile blood for M seconds; the mixing time M is in the range of 180 seconds or more; the mixing temperature is 18 degrees Celsius to 45 degrees Celsius.

[0025] One of the technical effects of the above-mentioned technical solution is that, through the cleverly designed hypotonic environment, the single dye component in the formula can quickly cross the cell membrane and enter the cell by taking advantage of the ion concentration difference inside and outside the cell, making it easy for the dye to enter the cell membrane and bind to the nuclear material inside the cell, thus completing the rapid staining of the cell nuclear material; and multiple different types of cells can be stained quickly with a single reagent addition, realizing single-step staining with a single dye staining reagent.

[0026] The second technical effect of the above-mentioned technical solution is that the aldehyde-containing substances in the staining stabilizer B10 react with the proteins on the cell membrane surface to form a colloidal polymer layer, thereby stabilizing and fixing the cell morphology, and forming fixed dye channels on the cell membrane surface. After the cells are fixed, the dye can more easily penetrate the cell membrane, achieving single-step rapid staining.

[0027] The third technical effect of the above-mentioned technical solution is that by combining a cleverly designed hypotonic environment with a pH of 7-8, the nuclei and nucleic acid substances of various cell types are more likely to exhibit negatively charged properties, making them particularly easy to combine with positively charged acidic or basic dyes. Therefore, only one positively charged acidic or basic dye can be used to stain different types of cells simultaneously. That is, nucleated red blood cells and neutrophils, eosinophils, basophils, lymphocytes, and monocytes in white blood cells can all be fully stained under this environmental condition, reaching a level sufficient to support the identification of various cell types under a bright-field microscope.

[0028] The fourth technical effect of the above-mentioned technical solution is that the positively charged dye in staining agent A10 can bind to the negatively charged phosphate groups in the cell. Based on the binding of electrical properties, the binding of the dye to the negatively charged nucleic acid groups in the cell nucleus is more stable, so that cell nuclei with different acid-base properties can be stained; and the staining effect can be maintained for a longer time.

[0029] The fifth technical effect of the above-mentioned technical solution is that the cleverly designed hypotonic environment keeps the osmotic pressure in a very delicate balance. During the staining process, the cells will swell slightly while maintaining their morphology. Within this osmotic pressure range, the slight swelling of the cells also increases the distinction between the cell nucleus, cell membrane, and other cytoplasm, which is more conducive to the identification and differentiation of different cell types.

[0030] The sixth technical effect of the above-mentioned technical solution: The neomethylene blue in staining agent A10 is positively charged. After entering the cell nucleus, it reduces the negative charge between phosphate groups and causes condensation; after condensation, the internal structure of the cell is more clearly visible. Attached Figure Description

[0031] Figure 1 This is a table showing the components of several examples and comparative examples of a single-dye staining reagent for single-step staining of multicellular blood in reptiles.

[0032] Figure 2 This is a collection of images of blood cells observed under a microscope after staining turtle blood cells according to Examples 1, 2, and 3.

[0033] Figure 3 This is a collection of images showing some of the blood cells in turtle blood cells after staining the sample in Example 1, magnified under a microscope. Detailed Implementation

[0034] The contents of this application will be further described in detail below with reference to the accompanying drawings. It should be noted that the following description is of preferred embodiments of the present invention and does not constitute any limitation on the present invention. The description of the preferred embodiments of the present invention is merely an explanation of the general principles of the invention. The use of terms such as "first," "second," and "A," "B," etc., in the present invention is for ease of explanation only and does not represent a temporal or spatial order.

[0035] like Figure 1 The table shows the components of several examples and comparative examples of a single-dye staining reagent for single-step staining of multicellular reptile blood. In one example of the formulation of the single-dye staining reagent for single-step staining of multicellular reptile blood, the reagent includes staining agent A10, osmotic buffer salt A30, staining stabilizer B10, and water; staining agent A10 is neomethylene blue; the concentration of neomethylene blue is 0.3 × 10⁻⁶. -4 ~3.0×10 -4g / ml; Osmotic buffer A30, used to provide a relatively low osmotic pressure relative to blood, with an osmotic pressure range of 170 mosmol / kg to 210 mosmol / kg; simultaneously, osmotic buffer A30 provides a weakly alkaline environment with a pH of 7-8; staining stabilizer B10 includes aldehydes, which chemically cross-link with proteins on the cell membrane surface, accelerating the entry of staining agent A10 into the cell. Figure 1 The table shown presents formulations for several single-dye staining reagents for single-step staining of multicellular blood in turtles, as well as formulations for single-dye staining reagents for single-step staining of multicellular blood in snakes and lizards.

[0036] Staining agent A10 comprises a positively charged basic dye that binds to negatively charged phosphate groups in cells, forming stained phosphate groups. The precise pH value and hypotonic environment allow for the staining of various cell types using a simple, single-dye, single-step reagent. It boasts high staining efficiency, completing staining in as little as 20 seconds, or even tens of seconds, compared to several minutes for dry slides. In existing technologies, neomethylene blue, a basic dye, is classified as a cationic dye, typically used to stain substances containing anionic chromophores in weakly acidic staining baths. Reagents using this dye for cell staining under weakly alkaline conditions are unprecedented. The formulation in this application overcomes this limitation, enabling this basic dye to achieve excellent staining results even in alkaline environments.

[0037] like Figure 1 As shown in the formulation examples of the single-dye staining reagent for multi-cell single-step staining of reptile blood, used for turtle blood cell staining, the pH value is 7.1 to 7.5; the osmotic buffer salt A30 includes disodium hydrogen phosphate and potassium dihydrogen phosphate. For turtle blood cell staining, the disodium hydrogen phosphate concentration is 4.3 × 10⁻⁶. -2 ~5.7×10 -2 The concentration of potassium dihydrogen phosphate is 9.5 × 10 mol / L. -3 ~2.5×10 -2 mol / L; pH value is 7.1~7.5.

[0038] like Figure 1 As shown in the formulation examples of the single-dye staining reagent for multi-cell single-step staining of reptile blood, the staining stabilizer B10 includes formaldehyde and glutaraldehyde. For turtle blood cell staining, the formaldehyde concentration is 0.9 × 10⁻⁶. -2 mol / L~1.3×10 -2 mol / L; glutaraldehyde concentration is 3.6 × 10⁻⁶ mol / L. -3 mol / L~4.8×10 -3mol / L. Osmotic buffer salt A30 also includes NaCl, with a concentration range of 4.67 × 10⁻⁶ mol / L. -3 mol / L~1.98×10 -2 mol / L.

[0039] In some embodiments not shown in the accompanying drawings, each 500 ml of staining reagent includes 0.5 mL of proClean950 solution, i.e., a preservative.

[0040] In an embodiment of a rapid single-step staining method for reptile blood cells not shown in some of the accompanying drawings, the above-described single-dye staining reagent for multi-cell single-step staining of reptile blood is used; the method includes a step of mixing a standard blood sample with the above-described single-dye staining reagent for multi-cell single-step staining of reptile blood for M seconds; the mixing time M is in the range of 180 seconds or more; the mixing temperature is 18°C ​​to 45°C. 25-45°C; 30°C is optimal. No external power supply is required for heating. It can be achieved at room temperature.

[0041] While the present invention has been described and illustrated with reference to preferred embodiments and several alternatives, the invention is not limited to the specific descriptions herein. Other alternatives or equivalent components may also be used to practice the invention.

Claims

1. A single-dye staining reagent for single-step staining of multicellular reptile blood, characterized in that, It includes dye A10, osmotic buffer salt A30, staining stabilizer B10 and water; The staining agent A10 is neomethylene blue; the concentration of neomethylene blue is 0.3 × 10⁻⁶. -4 ~3.0×10 -4 g / ml; Osmotic buffer salt A30 is used to provide a low osmotic pressure environment relative to blood, with an osmolar concentration range of 170 mosmol / kg to 210 mosmol / kg in the low osmotic pressure environment; Meanwhile, osmotic buffer salt A30 is used to provide a slightly alkaline environment with a pH of 7-8; Dye stabilizer B10 includes aldehyde-containing substances; Used for staining turtle blood cells, pH 7.1 to 7.5; Osmotic buffer salt A30 includes disodium hydrogen phosphate and potassium dihydrogen phosphate; The concentration of disodium hydrogen phosphate is 4.3 × 10⁻⁶. -2 ~5.7×10 -2 The concentration of potassium dihydrogen phosphate is 9.5 × 10 mol / L. -3 ~2.5×10 - 2 mol / L; pH value is 7.1~7.

5.

2. The single-dye staining reagent for multi-cell single-step staining of reptile blood according to claim 1, characterized in that, The dyeing stabilizer B10 includes formaldehyde and glutaraldehyde.

3. The single-dye staining reagent for multi-cell single-step staining of reptile blood according to claim 2, characterized in that, For staining turtle blood cells, the formaldehyde concentration was 0.9 × 10⁻⁶. -2 mol / L ~ 1.3 × 10 -2 mol / L; glutaraldehyde concentration is 3.6 × 10⁻⁶ mol / L. -3 mol / L ~ 4.8 × 10 -3 mol / L.

4. The single-dye staining reagent for multi-cell single-step staining of reptile blood according to claim 1, characterized in that, Osmotic buffer salt A30 also includes NaCl, with a concentration range of 4.67 × 10⁻⁶. -3 mol / L ~ 1.98 × 10 -2 mol / L.

5. The single-dye staining reagent for multi-cell single-step staining of reptile blood according to claim 1, characterized in that... Each 500ml of staining reagent contains 0.5mL of proClean950 solution, which is a preservative.

6. A rapid, single-step staining method for multi-celled blood of reptiles, characterized in that, Use the single-dye staining reagent for multi-cell single-step staining of reptile blood as described in any one of claims 1 to 5; This includes the step of mixing a standard blood sample with the single-dye staining reagent for multi-cell single-step staining of reptile blood for M seconds; The mixing time M is in the range of 180 seconds or more; the mixing temperature is 18 degrees Celsius to 45 degrees Celsius.

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