CN117990471A - Cell stain and application thereof in virus TCID50 determination - Google Patents
Cell stain and application thereof in virus TCID50 determination Download PDFInfo
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Abstract
The invention provides a cell stain and application thereof in virus TCID50 determination. The cell stain of the present invention comprises the following raw materials: 3-8 g/L of Rui pigment, 0.3-0.8 g/L of Giemsa pigment, 0.3-0.8 g/L of methylene blue, 10-15 g/L of anhydrous calcium chloride, 3-8 g/L of anhydrous potassium dihydrogen phosphate, 0.3-0.8 g/L of anhydrous disodium hydrogen phosphate, 20-35 mL/L of surfactant and the balance of solvent, wherein the solvent is a composition of dimethyl sulfoxide, glycerol and methanol, and the volume ratio of dimethyl sulfoxide, glycerol and methanol is 1: (1-5): (25-35). The cell stain provided by the invention has good staining effect on the adherence indication cells in the measurement of the virus TCID50, does not cause a large number of cell lysis, does not need to fix cells during staining, and can avoid the risk of cell shedding, thereby improving the accuracy of the detection result of the virus TCID 50.
Description
Technical Field
The invention belongs to the technical field of cell staining, and particularly relates to a cell stain and application thereof in virus TCID50 determination.
Background
Virus removal/inactivation verification the viral removal/inactivation capacity of a process step is generally assessed by adding an indicator of the virus. The virus titer (VIRAL TITER) refers to the content of a live virus or a functional virus in a unit volume, and is closely related to the pathogenic capacity of the virus, so that accurate measurement of the titer of a specific virus is important for prevention, control and elimination of virus infection, and is one of the necessary detection procedures for quantitatively amplifying and indicating the virus. Viral titers are typically expressed as tissue half-infectious end-point titration TCID50 (50% tissue culture infective dose), i.e., the minimum amount of virus required to cause half-cytopathic effects (cytopathic effect, CPE) in cell culture plate wells or tubes over a specified period of time. Currently, methods for detecting virus titer are plaque method (Virus plaque formation), real-time fluorescent quantitative PCR method (Quantitative Real-time PCR, qPCR), immunocytochemistry method (Immunocytochemistry, ICC), enzyme-linked immunosorbent assay (Enzyme-Linked ImmunoSorbent Assay, ELISA), transmission electron microscopy (Transmission electron microscopes, TEM), high-performance liquid chromatography (High-performance liquid chromatography, HPLC), etc. However, the TCID50 method is still the most commonly used method, and has advantages of lower cost, fewer steps, simpler calculation and the like compared with other methods, but also has some defects, such as visual fatigue possibly generated when the cells under the optical microscope are observed with naked eyes for a long time, so as to generate subjective observation errors; in addition, it is clear that the transparent indicator cell itself is highly required for optical microscope imaging in terms of its morphology and relationship with surrounding cells.
In this regard, patent CN201611194249.6 changes Cytopathy (CPE), originally observed by naked eye through an optical microscope, into a binding with anti-porcine parvovirus antibody based on the conventional TCID50, and the color development is catalyzed by horseradish peroxidase and the absorbance value is detected. But adds a great deal of unnecessary procedures, reagent purchasing and preserving costs in the process; in addition, since antibodies have specificity, the method has limited virus types which can be detected, has poor universality, and when other types of viruses are detected, the antibodies need to be prepared again, thereby further increasing the cost. The patent CN202111244738.9 combines crystal violet staining with TCID50 and stains cells with crystal violet prior to viewing the cells, thereby darkening the cells and making it easier to observe the results than traditional TCID 50. However, the method has the defects that the experiment result is inaccurate due to the fact that the dyeing degree of crystal violet is deeper and cell falling in holes is easy to occur in the processes of cell fixation and cleaning of the dyeing liquid. Patent cn201610522012.X combines TCID50 with trypan blue staining method, and in theory trypan blue only can enter dead cells but not stain living cells, but in actual operation process, pigment deposition can be generated on the periphery of living cells when the cell density is too high, and sensitive cells cultured by TCID50 method just contact each other, finally resulting in too deep background color, inconvenient observation and difficult distinction of living cells from dead cells. In addition, CPE is not apparent at very early and early stages of virus infection, and does not cause cell death, and trypan blue cannot enter cells for color development, so that the efficacy of viruses is underestimated, systematic errors are caused, and even virus infection accidents occur.
Therefore, in order to reduce the difficulty of cytopathic observation and improve the accuracy of the measurement result of the virus TCID50, it is still necessary to develop a staining method and a matched cell stain suitable for the indicator cells in the measurement of the virus TCID 50.
Disclosure of Invention
The invention aims to solve the problem of providing a cell stain which has good staining effect on an adherence indication cell in the measurement of virus TCID50 and can improve the accuracy of the detection result of the virus TCID50, and further provides a preparation method and application of the cell stain.
In order to solve the technical problems, the invention adopts the following technical scheme:
The first aspect of the present invention provides a cell stain, the cell stain comprising the following raw materials: 3-8 g/L of Rui pigment, 0.3-0.8 g/L of Giemsa pigment, 0.3-0.8 g/L of methylene blue, 10-15 g/L of anhydrous calcium chloride, 3-8 g/L of anhydrous potassium dihydrogen phosphate, 0.3-0.8 g/L of anhydrous disodium hydrogen phosphate, 20-35 mL/L of surfactant and the balance of solvent, wherein the solvent is a composition of dimethyl sulfoxide, glycerol and methanol, and the volume ratio of the dimethyl sulfoxide, the glycerol and the methanol is 1: (1-5): (25-35).
Further preferably, the raw material composition of the cell stain includes: 4-6 g/L of Rui pigment, 0.4-0.6 g/L of Giemsa pigment, 0.4-0.6 g/L of methylene blue, 10-12 g/L of anhydrous calcium chloride, 4-6 g/L of anhydrous potassium dihydrogen phosphate, 0.4-0.6 g/L of anhydrous disodium hydrogen phosphate, 20-30 mL/L of surfactant and the balance of solvent, wherein the solvent is a composition of dimethyl sulfoxide, glycerol and methanol, and the volume ratio of the dimethyl sulfoxide, the glycerol and the methanol is 1: (1-5): (30-35).
Further preferably, the surfactant is one or more of tween-20, tween-40, tween-80, triton X100 or triton X114.
The second aspect of the present invention provides a method for preparing the above cell stain, comprising the steps of:
(1) Mixing and grinding the Rui's pigment, jim's pigment, methylene blue, anhydrous calcium chloride, anhydrous potassium dihydrogen phosphate and anhydrous disodium hydrogen phosphate into powder, and sieving the powder with a 100-mesh sieve;
(2) Mixing and grinding part of the surfactant, part of dimethyl sulfoxide, glycerol and methanol with the powder for 5-15 min, standing for 15-25 min, and removing the upper liquid; mixing and grinding the lower layer precipitate, part of the surfactant, part of dimethyl sulfoxide, glycerol and methanol for 5-15 min, standing for 15-25 min, removing the upper layer liquid, and repeating for 2-5 times; and combining the upper layer liquid, and standing at room temperature in a dark place for at least 24 hours to obtain the cell stain.
In a third aspect, the invention provides the use of the above cell stain in adherent cell staining.
In a fourth aspect, the invention provides the use of a cell stain as described above in a viral TCID50 assay, wherein the cell stain is used to stain indicator cells in a viral titer TCID50 assay, the indicator cells being adherent cells.
Preferably, the adherent cells are mouse subcutaneous connective tissue cells (A9 cells), african green monkey kidney cells (Vero cells), chinese hamster ovary cells (CHO cells), mouse embryo fibroblasts (NIH-3T 3 cells), hamster kidney fibroblasts (BHK-21 cells) or mouse skin fibroblasts (Murray Denne cells Mus dunni cells).
In a fifth aspect, the present invention provides a method for staining indicator cells in a TCID50 assay, wherein the above cell stain is added to indicator cells cultured in a medium containing no virus and indicator cells cultured in a medium containing virus, the cells are left to stand at room temperature for 8 to 12 minutes, the cell stain is removed, the plate is washed with sterile water, and the indicator cells are dried in the air, and no fixing operation is required before staining, and the indicator cells are adherent cells.
The sixth aspect of the present invention provides a method for determining TCID50 of a virus, the method for determining TCID50 of a virus comprising the steps of:
(1) Inoculating indicator cells into cell culture holes of a cell culture plate, and culturing the indicator cells by adopting a DMEM complete culture solution until the indicator cells are plated, wherein the indicator cells are adherent cells;
(2) Performing gradient dilution on the virus stock solution by adopting a DMEM complete culture solution to obtain virus diluents with different dilutions, replacing the culture medium in the cell culture hole in the step (1) with the virus diluents, continuously culturing for 6-8 days, and replacing the virus diluents in the cell culture hole with the DMEM complete culture solution when culturing for 3-5 days;
(3) Replacing the DMEM complete culture solution in the cell culture hole in the step (2) with the cell stain, standing and staining at room temperature for 8-12 min, removing the cell stain, washing the plate with sterile water, airing, observing and indicating cell morphology under an optical microscope, counting cytopathy, and calculating the virus titer.
Preferably, the indicator cell is a mouse subcutaneous connective tissue cell (A9 cell), an African green monkey kidney cell (Vero cell), a Chinese hamster ovary cell (CHO cell), a mouse embryo fibroblast cell (NIH-3T 3 cell), a hamster kidney fibroblast cell (BHK-21 cell) or a mouse skin fibroblast cell (Murray Denne cell Mus dunni cell).
Compared with the prior art, the invention has the following advantages:
The cell stain has good staining effect on the adherent indication cells in the measurement of the virus TCID50, can clearly display the cell morphology, is convenient for counting cytopathy, has small influence on cells, does not cause a large number of cell lysis, does not need to fix the cells when being used for staining, simplifies the operation steps, shortens the operation time, and can also avoid the risk of cell shedding, thereby improving the accuracy of the measurement result of the virus TCID 50.
Drawings
FIG. 1 is a staining microscopic image of negative control A9 cells without virus infection in example 1;
FIG. 2 is a staining microscopic image of the infection of A9 cells with MVM virus solution of 10 -6 dilution in example 1;
FIG. 3 is a staining microscopic image of the A9 cells infected with MVM virus solution of 10 -3 dilution in example 1;
FIG. 4 is a staining microscopic image of negative control A9 cells without virus infection in comparative example 1;
FIG. 5 is a staining microscopic image of the A9 cells infected with MVM virus solution of 10 -6 dilution in comparative example 1;
FIG. 6 is a staining microscopic image of the A9 cells infected with MVM virus solution of 10 -3 dilution in comparative example 1;
FIG. 7 is a staining microscopic image of negative control A9 cells without virus infection in comparative example 2;
FIG. 8 is a staining microscopic image of the A9 cells infected with MVM virus solution of 10 -6 dilution in comparative example 2;
FIG. 9 is a staining microscopic image of the A9 cells infected with MVM virus solution of 10 -3 dilution in comparative example 2;
FIG. 10 is a staining microscopic image of A9 cells infected with MVM virus solution of 10 -5 dilution in comparative example 3, commercially available Giemsa staining solution kit brand A;
FIG. 11 is a staining microscopic image of A9 cells infected with MVM virus solution of comparative example 3 at a dilution of 10 -5, commercially available Giemsa staining solution kit brand B;
FIG. 12 is a staining microscopic image of the A9 cells infected with MVM virus solution of 10 -5 dilution in comparative example 4;
FIG. 13 is a staining microscopic image of the A9 cells infected with MVM virus solution of 10 -5 dilution in comparative example 5;
FIG. 14 is a staining microscopic image of the A9 cells infected with MVM virus solution of 10 -5 dilution in comparative example 6;
FIG. 15 is a staining microscopic image of the A9 cells infected with MVM virus solution of 10 -5 dilution in comparative example 7;
FIG. 16 is a staining microscopic image of the negative control Vero cells without virus infection in example 2;
FIG. 17 is a staining microscopic image of the infection of Vero cells with EMCV virus solution of 10 -6 dilution in example 2;
FIG. 18 is a staining microscopic image of the infection of Vero cells with EMCV virus solution of 10 -3 dilution in example 2.
Detailed Description
When the virus TCID50 method is used for measuring the virus titer, a inspector is required to inspect and observe the condition of the indicated cytopathy, when transparent cells are observed, visual fatigue is easy to generate, systematic errors are caused, and the dependency on the working experience of technicians is high. Although the observation difficulty can be reduced by staining the indicator cells, in the experiment, it is found that the judgment of cytopathy is affected by too deep staining or that cytopathy cannot be clearly distinguished due to cell lysis or that the number of cells is lost due to cell fixation, plate washing and other operations, and the like, which all result in inaccurate detection results of the virus TCID 50. In order to solve these problems, the inventors have conducted a great deal of research and experiments on the selection, improvement and staining method of a stain in combination with the requirements of the detection of the virus TCID50, thereby proposing a cell stain which has a good staining effect on the adherent indicator cells in the measurement of the virus TCID50 and can improve the accuracy of the detection result of the virus TCID50, and a method for measuring the virus TCID50 in combination with the staining method of the stain.
Specifically, the cell stain of the present invention comprises the following raw materials: 3-8 g/L of Rui pigment, 0.3-0.8 g/L of Giemsa pigment, 0.3-0.8 g/L of methylene blue, 10-15 g/L of anhydrous calcium chloride, 3-8 g/L of anhydrous potassium dihydrogen phosphate, 0.3-0.8 g/L of anhydrous disodium hydrogen phosphate, 20-35 mL/L of surfactant and the balance of solvent, wherein the solvent is a composition of dimethyl sulfoxide, glycerol and methanol, and the volume ratio of dimethyl sulfoxide, glycerol and methanol is 1: (1-5): (25-35). According to the invention, the formula and the preparation method of the existing Switzerland giemsa dye liquor are adjusted and optimized, the obtained cell stain has good staining effect on the adherent indication cells in the virus TCID50 measurement, a large number of cells are not cracked, and meanwhile, the cell fixation is not needed during staining, so that the risk of cell shedding can be avoided, and the accuracy of the measurement result of the virus TCID50 is improved.
Further, the method for measuring the TCID50 of a virus indicating cell staining by using the cell stain of the present invention comprises the steps of:
(1) Inoculating indicator cells into cell culture holes of a cell culture plate, and culturing by adopting DMEM complete culture solution until the indicator cells are plated, wherein the indicator cells are adherent cells;
(2) Carrying out gradient dilution on the virus stock solution by adopting a DMEM complete culture solution to obtain virus diluents with different dilutions, replacing the culture medium in the cell culture hole in the step (1) with the virus diluents, and continuously culturing for 3-8 days, wherein the virus diluents in the cell culture hole can be replaced with the DMEM complete culture solution selectively when culturing for 3-5 days;
(3) Replacing the DMEM complete culture solution in the cell culture hole in the step (2) with the cell stain, standing and staining at room temperature for 8-12 min, removing the cell stain, washing the plate with sterile water, airing, observing and indicating cell morphology under an optical microscope, counting cytopathy, and calculating the virus titer.
The cell stain is an improved Giemsa staining solution designed by the inventor, a fixing step is not needed in the staining process, and the cell stain can be directly added for staining, so that compared with the existing staining method, the method has the advantages of simplifying the steps, shortening the staining time and avoiding the cell shedding of experimenters due to operation errors to a certain extent. The cell stain can dye the cytoplasm into red, dye the nucleus into blue-violet, reduce the difficulty of observing cell morphology, and is favorable for observing early pathological changes of cells.
The invention is further described below with reference to examples. The present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions which are not noted are conventional conditions in the industry. The technical features of the various embodiments of the present invention may be combined with each other as long as they do not collide with each other.
The culture conditions referred to in the following examples and comparative examples were: 37 ℃,5% co 2.
In the following examples and comparative examples, the Rayleigh pigment, gimssa pigment, methylene blue, anhydrous calcium chloride, anhydrous potassium dihydrogen phosphate and anhydrous disodium hydrogen phosphate were all anhydrous products or products dried to constant weight, and the amounts described were all dry weights.
In the following examples and comparative examples, DMEM complete broth was purchased from Shanghai Ao Pu Mai Biotech Co., ltd. (cat. No. P081702-001); richter pigment is available from Beijing Soy Bao technology Co., ltd. (cat# W8010); giemsa pigment is available from Beijing Soy Bao technology Co., ltd. (cat# G8220); methylene blue was purchased from Beijing Soy Bao technology Co., ltd. (cat# M8030); the crystal violet dye liquor was purchased from Beijing Soy Bao technology Co., ltd. (cat# G1071); trypan blue staining solution was purchased from Shanghai Biotechnology Co., ltd. (cat number: E607338); commercial Jiemsa staining solution kit brand A was purchased from Beijing Boaosen Biotechnology Co., ltd (Bioss organism) and brand B was purchased from Beijing white shark easy technology Co., ltd (Biosharp organism). The remaining raw materials or reagents not specifically described are commercially available products conventionally used in the art.
The indicated cytoscopies of the following examples and comparative examples were each observed under a 20 x objective lens.
Example 1
The present example provides a method for staining indicator cells (A9 cells) in MVM virus titer TCID50 assay, specifically as follows:
1. MVM virus infection A9 cells: a9 cells were seeded in 96-well cell culture plates at a number of 1×10 4 cells per well and cultured for 24h using DMEM complete broth. The MVM virus stock was serially diluted 10-fold in gradient from 10 -1 to 10 -8 using DMEM complete medium. The medium in the cell culture wells of the 96-well cell culture plate was blotted to obtain plated A9 cells, and then MVM virus dilutions of different dilutions were added to the plated A9 cells, respectively, while negative controls were set to the plated A9 cells to which DMEM complete culture solution without MVM virus was added, each treatment being set to 6 replicates. Culturing was continued for 7 days, and DMEM complete medium was changed once from day 4.
2. Indicator cell A9 cell staining:
After the MVM virus infects A9 cells and negative control cells and cultures for 7 days, the culture medium in the cell culture hole is removed, self-made cell coloring agent is added to fully cover the bottom of the cell culture hole, the cell culture hole is stained for 10min at room temperature, a 96-well plate is washed by sterile water, the morphology of the A9 cells is observed under an optical microscope after the cell culture hole is dried in a fume hood, the cell culture hole with Cytopathy (CPE) is recorded, and the virus titer is calculated.
The preparation method of the cell stain used in this example is:
(1) Weighing 5g of Rui's pigment, 0.5g of Giemsa's pigment, 0.5g of methylene blue, 11g of anhydrous calcium chloride, 5g of anhydrous potassium dihydrogen phosphate, 0.5g of anhydrous disodium hydrogen phosphate, 25mL of Tween-20, 25mL of dimethyl sulfoxide, 50mL of glycerol and 800mL of methanol;
(2) Putting the Rui pigment, the Jim Sapigment, the methylene blue, the anhydrous calcium chloride, the anhydrous potassium dihydrogen phosphate and the anhydrous disodium hydrogen phosphate into a mortar, lightly tapping the mortar with a milk stick, grinding the mortar into fine powder, and sieving the powder with a 100-mesh sieve;
(3) Adding 6.25mL of Tween-20, 6.25mL of dimethyl sulfoxide, 12.5mL of glycerol and 200mL of methanol, and grinding for 10min;
(4) Covering with a preservative film to prevent methanol volatilization and precipitation for 20min, and taking out the upper layer dye liquor by using a rubber head suction pipe;
(5) And (3) circularly performing the steps (3) and (4) until the Tween-20, the dimethyl sulfoxide, the glycerol and the methanol are completely used up, transferring all the collected upper layer dye liquor into a reagent bottle, and standing for 24 hours at room temperature in a dark place for use or continuing to preserve in a dark place.
The microscopic examination results of the embodiment show that the cytoplasm and the cell nucleus structure (fig. 1-3) can be clearly observed under the condition of no virus and virus infection with different dilution factors, so that cytopathy after virus infection can be conveniently confirmed, and the virus titer of the MVM virus stock solution can be calculated according to the microscopic examination results.
Comparative example 1
This comparative example provides a crystal violet staining method for indicator cell A9 cells in MVM virus titer TCID50 assay, specifically as follows:
1. MVM virus infection A9 cells: as in example 1.
2. Indicator cell A9 cell staining: after the MVM virus infects the A9 cells and the negative control cells to culture for 7 days, removing the culture medium in the cell culture holes, adding methanol to fully cover the bottoms of the cell holes, and fixing the cells for 15min; sucking the methanol by using a pipette, and drying at room temperature for 10min to volatilize the methanol; adding crystal violet dye solution which is diluted to 2% of working concentration by PBS in advance, fully covering the bottom of a cell hole, and dyeing for 10min at room temperature; the 96-well plate was washed with sterile water, dried in a fume hood, and the morphology of the A9 cells was observed under an optical microscope.
The microscopic examination results of the embodiment show that under the condition of no virus and different dilution factors, the staining of the indication cells is too deep (fig. 4-6), especially when the dilution factors of the virus are higher, the cell density is high, the cell structure is difficult to observe, and therefore, cytopathy is difficult to distinguish, and the detection result is inaccurate.
Comparative example 2
This comparative example provides a trypan blue staining method for indicator cell A9 cells in MVM virus titer TCID50 assay, specifically as follows:
1. MVM virus infection A9 cells: as in example 1.
2. Indicator cell A9 cell staining: after the MVM virus infects the A9 cells and the negative control cells to culture for 7 days, removing the culture medium in the cell culture holes, adding methanol to fully cover the bottoms of the cell holes, and fixing the cells for 15min; sucking the methanol by using a pipette, and drying at room temperature for 10min to volatilize the methanol; adding trypan blue staining solution which is diluted to 0.04% working concentration by PBS in advance, fully covering the bottom of a cell hole, and staining for 3min at room temperature; the 96-well plate was washed with sterile water, dried in a fume hood, and the morphology of the A9 cells was observed under an optical microscope.
The microscopic examination results of the comparative example show that the cell density is high without virus and at high dilution factor, and the cell structure is difficult to observe (fig. 7-9), so that cytopathy is difficult to distinguish, and the detection result is inaccurate.
Comparative example 3
The indicator cells A9 were stained using two commercially available giemsa staining solution kits (designated as brand a, purchased from beijing boaosen biotechnology limited, and designated as brand B, purchased from beijing white shark easy technology limited) according to the instructions of the corresponding commercially available giemsa staining solution kit.
Brand a:
1. MVM virus infection A9 cells: as in example 1.
2. Indicator cell A9 cell staining: after the MVM virus infects the A9 cells and the negative control cells to culture for 7 days, removing the culture medium in the cell culture holes, adding methanol to fully cover the bottoms of the cell holes, and fixing the cells for 15min; sucking the methanol by using a pipette, and drying at room temperature for 10min to volatilize the methanol; adding a staining working solution prepared by mixing a Giemsa staining solution (component A) and a phosphoric acid buffer solution (component B) in equal proportion, fully covering the bottom of a cell hole, and staining for 10min at room temperature; the 96-well plate was washed with sterile water, and after air-dried in a fume hood, the staining effect of A9 cells was observed under an optical microscope, and the Giemsa staining solution of brand A had only good staining ability on cytoplasm and had poor staining effect on nuclei (FIG. 10), and the detection accuracy was low when observing cytopathy after virus infection.
Brand B:
1. MVM virus infection A9 cells: as in example 1.
2. Indicator cell A9 cell staining: after the MVM virus infects A9 cells and negative control cells and cultures for 7 days, after the cells are cultured for 7 days, removing the culture medium in the cell culture holes, adding methanol to fully cover the bottoms of the cell holes, and fixing the cells for 15min; sucking the methanol by using a pipette, and drying at room temperature for 10min to volatilize the methanol; adding Giemsa staining solution to fully cover the bottom of the cell hole, and staining at room temperature for 10min; the 96-well plate was washed with sterile water, and after air-dried in a fume hood, the morphology of A9 cells was observed under an optical microscope, which also had a good staining ability for cytoplasm and poor staining effect for nuclei (fig. 11).
Experiments show that methanol is necessary to fix cells during staining of the brand A and the brand B in the comparative example, otherwise, the cell nucleus is not obvious in staining, cytopathy cannot be accurately distinguished, and the detection result is inaccurate. And still need to suck the methyl alcohol after carrying out methyl alcohol fixed cell, need very careful during the operation, misjudgement that the misjudgement of testing result caused by the improper operation causes the cell to drop when preventing to suck the methyl alcohol, complex operation and time-consuming.
The comparison conditions of the operation duration, difficulty, cost and the like of the different dyeing methods in the example 1 and the comparative examples 1-3 are shown in the table 1.
Note that: difficulty and cost are indicated by "+", which represents ease/cost; "++" represents moderate difficulty/moderate cost; "+". ++'s representative of complex/costly.
Comparative example 4
This comparative example provides another method for staining indicator cells (A9 cells) in an MVM virus titer TCID50 assay, which is operated substantially the same as example 1, except that the cell stain is made in a homemade manner.
The preparation method of the cell dye solution adopted in the comparative example comprises the following steps:
(1) Weighing 5g of Rui's pigment, 0.5g of Giemsa pigment, 0.5g of methylene blue, 5g of anhydrous potassium dihydrogen phosphate, 0.5g of anhydrous disodium hydrogen phosphate, weighing 25mL of Tween-20, 50mL of glycerol and 800mL of methanol;
(2) Putting the Rui's pigment, jim's pigment, methylene blue, anhydrous potassium dihydrogen phosphate and anhydrous disodium hydrogen phosphate into a mortar, lightly tapping the mortar with a milk stick, grinding the mortar into fine powder, and sieving the powder with a 100-mesh sieve;
(3) Adding 6.25mL of Tween-20, 12.5mL of glycerol and 200mL of methanol, and grinding for 10min;
(4) Covering with a preservative film to prevent methanol volatilization and precipitation for 20min, and taking out the upper layer dye liquor by using a rubber head suction pipe;
(5) And (3) circularly performing the steps (3) and (4) until the Tween-20, the glycerol and the methanol are completely used up, transferring all the collected upper layer dye liquor into a reagent bottle, and standing for 24 hours at room temperature in a dark place for use or continuing to preserve in a dark place.
The staining effect of this comparative example was superior to comparative examples 1 to 3, but not as good as that of example 1, mainly cytoplasmic staining effect was inferior to that of example 1 (fig. 12).
Comparative example 5
This comparative example provides another method for staining indicator cells (A9 cells) in an MVM virus titer TCID50 assay, which is operated substantially the same as example 1, except that the cell stain is made in a homemade manner.
The preparation method of the cell dye solution adopted in the comparative example comprises the following steps:
(1) Weighing 5g of Rui's pigment, 0.5g of Giemsa's pigment, 0.5g of methylene blue, 11g of anhydrous calcium chloride, 5g of anhydrous potassium dihydrogen phosphate, 0.5g of anhydrous disodium hydrogen phosphate, weighing 25mL of Tween-20, 50mL of glycerol and 800mL of methanol;
(2) Putting the Rui pigment, the Jim Sapigment, the methylene blue, the anhydrous calcium chloride, the anhydrous potassium dihydrogen phosphate and the anhydrous disodium hydrogen phosphate into a mortar, lightly tapping the mortar with a milk stick, grinding the mortar into fine powder, and sieving the powder with a 100-mesh sieve;
(3) Adding 6.25mL of Tween-20, 12.5mL of glycerol and 200mL of methanol, and grinding for 10min;
(4) Covering with a preservative film to prevent methanol volatilization and precipitation for 20min, and taking out the upper layer dye liquor by using a rubber head suction pipe;
(5) And (3) circularly performing the steps (3) and (4) until the Tween-20, the glycerol and the methanol are completely used up, transferring all the collected upper layer dye liquor into a reagent bottle, and standing for 24 hours at room temperature in a dark place for use or continuing to preserve in a dark place.
The staining effect of this comparative example was superior to that of comparative examples 1 to 3, but not as good as that of example 1, and the same effect of staining mainly cytoplasm was inferior to that of example 1 (FIG. 13).
Comparative example 6
This comparative example provides another method for staining indicator cells (A9 cells) in an MVM virus titer TCID50 assay, which is operated substantially the same as example 1, except that the cell stain is made in a homemade manner.
The preparation method of the cell dye solution adopted in the comparative example comprises the following steps:
(1) Weighing 5g of Rui's pigment, 0.5g of Giemsa's pigment, 0.5g of methylene blue, 15g of anhydrous calcium chloride, 5g of anhydrous potassium dihydrogen phosphate, 0.5g of anhydrous disodium hydrogen phosphate, weighing 25mL of Tween-20, 200mL of dimethyl sulfoxide, 100mL of glycerol and 500mL of methanol;
(2) Putting the Rui pigment, the Jim Sapigment, the methylene blue, the anhydrous calcium chloride, the anhydrous potassium dihydrogen phosphate and the anhydrous disodium hydrogen phosphate into a mortar, lightly tapping the mortar with a milk stick, grinding the mortar into fine powder, and sieving the powder with a 100-mesh sieve;
(3) Adding 6.25mL of Tween-20, 50mL of dimethyl sulfoxide, 25mL of glycerol and 125mL of methanol, and grinding for 10min;
(4) Covering with a preservative film to prevent methanol volatilization and precipitation for 20min, and taking out the upper layer dye liquor by using a rubber head suction pipe;
(5) And (3) circularly performing the steps (3) and (4) until the Tween-20, the glycerol and the methanol are completely used up, transferring all the collected upper layer dye liquor into a reagent bottle, and standing for 24 hours at room temperature in a dark place for use or continuing to preserve in a dark place.
More cell lysis occurred after staining of this comparative example, and cytopathy after virus infection could not be clearly distinguished (fig. 14).
Comparative example 7
This comparative example provides another method for staining indicator cells (A9 cells) in an MVM virus titer TCID50 assay, which is operated substantially the same as example 1, except that the cell stain is made in a homemade manner.
The preparation method of the cell dye solution adopted in the comparative example comprises the following steps:
(1) Weighing 5g of Rui's pigment, 0.5g of Giemsa's pigment, 0.5g of methylene blue, 5g of anhydrous calcium chloride, 5g of anhydrous potassium dihydrogen phosphate, 0.5g of anhydrous disodium hydrogen phosphate, weighing 25mL of Tween-20, 200mL of dimethyl sulfoxide and 800mL of methanol;
(2) Putting the Rui pigment, the Jim Sapigment, the methylene blue, the anhydrous calcium chloride, the anhydrous potassium dihydrogen phosphate and the anhydrous disodium hydrogen phosphate into a mortar, lightly tapping the mortar with a milk stick, grinding the mortar into fine powder, and sieving the powder with a 100-mesh sieve;
(3) Adding 6.25mL of Tween-20, 50mL of dimethyl sulfoxide and 200mL of methanol, and grinding for 10min;
(4) Covering with a preservative film to prevent methanol volatilization and precipitation for 20min, and taking out the upper layer dye liquor by using a rubber head suction pipe;
(5) And (3) circularly performing the steps (3) and (4) until the Tween-20, the dimethyl sulfoxide and the methanol are completely used up, transferring all the collected upper layer dye liquor into a reagent bottle, and standing for 24 hours at room temperature in a dark place for use or continuing to preserve in a dark place.
More cell lysis occurred after staining of this comparative example, and cytopathy after virus infection could not be clearly distinguished (fig. 15).
Example 2
The embodiment provides a staining method of indicator cell Vero cells in the measurement of EMCV virus titer TCID50, which is specifically as follows:
1. EMCV virus infection of Vero cells: vero cells were seeded in 96-well cell culture plates at a number of 1X 10 4 cells per well and cultured for 24h using DMEM complete medium. The MVM virus stock was serially diluted 10-fold in gradient from 10 -1 to 10 -6 using DMEM complete medium. The culture medium in the cell culture wells of the 96-well cell culture plate was drawn out to obtain plated Vero cells, and then EMCV virus dilutions of different dilutions were added to the plated Vero cells, respectively, while negative controls were set to the plated Vero cells to which DMEM complete culture solution without EMCV virus was added, 6 replicates were set for each treatment, and cultured for 3 days.
2. Indicator cell Vero cell staining:
after the Vero cells and the negative control cells are infected by EMCV virus and cultured for 3 days, the culture medium in the cell culture holes is removed, self-made cell coloring agent is added until the bottom of the cell culture holes is fully covered, the cells are stained for 10min at room temperature, a 96-well plate is washed by sterile water, the morphology of the Vero cells is observed under an optical microscope after the cells are dried in a fume hood, the cell culture holes with Cytopathy (CPE) are recorded, and the virus titer is calculated.
The homemade cell stain in this example was the same as in example 1.
The microscopic examination results of the embodiment show that the cytoplasm and the cell nucleus structure can be clearly observed under the condition of no virus and different dilution factors (fig. 16-18), so that cytopathy after virus infection can be conveniently confirmed, and the virus titer of the EMCV stock solution can be calculated according to the microscopic examination results. The staining method provided by the embodiment is suitable for staining the indicator cells in the measurement of the virus titer TCID50 of different viruses, and has wide applicability to a certain extent.
The present invention has been described in detail with the purpose of enabling those skilled in the art to understand the contents of the present invention and to implement the same, but not to limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.
Claims (10)
1. A cell stain, characterized in that: the cell stain comprises the following raw materials: 3-8 g/L of Rui pigment, 0.3-0.8 g/L of Giemsa pigment, 0.3-0.8 g/L of methylene blue, 10-15 g/L of anhydrous calcium chloride, 3-8 g/L of anhydrous potassium dihydrogen phosphate, 0.3-0.8 g/L of anhydrous disodium hydrogen phosphate, 20-35 mL/L of surfactant and the balance of solvent, wherein the solvent is a composition of dimethyl sulfoxide, glycerol and methanol, and the volume ratio of the dimethyl sulfoxide, the glycerol and the methanol is 1: (1-5): (25-35).
2. The cell stain of claim 1, wherein: the cell stain comprises the following raw materials: 4-6 g/L of Rui pigment, 0.4-0.6 g/L of Giemsa pigment, 0.4-0.6 g/L of methylene blue, 10-12 g/L of anhydrous calcium chloride, 4-6 g/L of anhydrous potassium dihydrogen phosphate, 0.4-0.6 g/L of anhydrous disodium hydrogen phosphate, 20-30 mL/L of surfactant and the balance of solvent, wherein the solvent is a composition of dimethyl sulfoxide, glycerol and methanol, and the volume ratio of the dimethyl sulfoxide, the glycerol and the methanol is 1: (1-5): (30-35).
3. The cell stain of claim 1, wherein: the surfactant is one or more of Tween-20, tween-40, tween-80, triton X100 or triton X114.
4. A method of preparing a cell stain according to any one of claims 1 to 3, wherein: the preparation method comprises the following steps:
(1) Mixing and grinding the Rui's pigment, jim's pigment, methylene blue, anhydrous calcium chloride, anhydrous potassium dihydrogen phosphate and anhydrous disodium hydrogen phosphate into powder, and sieving the powder with a 100-mesh sieve;
(2) Mixing and grinding part of the surfactant, part of dimethyl sulfoxide, glycerol and methanol with the powder for 5-15 min, standing for 15-25 min, and removing the upper liquid; mixing and grinding the lower layer precipitate, part of the surfactant, part of dimethyl sulfoxide, glycerol and methanol for 5-15 min, standing for 15-25 min, removing the upper layer liquid, and repeating for 2-5 times; and combining the upper layer liquid, and standing at room temperature in a dark place for at least 24 hours to obtain the cell stain.
5. Use of a cell stain according to any one of claims 1 to 3 for adherent cell staining.
6. Use of a cell stain according to any one of claims 1 to 3 in a virus TCID50 assay, wherein: and (3) staining indicator cells in the virus titer TCID50 measurement by using the cell staining agent, wherein the indicator cells are adherent cells.
7. The use according to claim 6, characterized in that: the adherent cells are mouse subcutaneous connective tissue cells, african green monkey kidney cells, chinese hamster ovary cells, mouse embryo fibroblasts, hamster kidney fibroblasts or mouse skin fibroblasts.
8. A method for staining indicator cells in a viral TCID50 assay, comprising: adding the cell stain according to any one of claims 1 to 3 to indicator cells cultured in a virus-free medium and indicator cells cultured in a virus-containing medium, standing and staining at room temperature for 8-12 min, removing the cell stain, washing the plates with sterile water, and air-drying, wherein the indicator cells do not need to be subjected to fixing operation before staining, and the indicator cells are adherent cells.
9. A method for determining the TCID50 of a virus, comprising the steps of: the virus TCID50 determination method comprises the following steps:
(1) Inoculating indicator cells into cell culture holes of a cell culture plate, and culturing the indicator cells by adopting a DMEM complete culture solution until the indicator cells are plated, wherein the indicator cells are adherent cells;
(2) Carrying out gradient dilution on the virus stock solution by adopting a DMEM complete culture solution to obtain virus diluents with different dilutions, replacing the culture medium in the cell culture hole in the step (1) with the virus diluents, and continuously culturing for 3-8 days, wherein the virus diluents in the cell culture hole can be replaced with the DMEM complete culture solution selectively when culturing for 3-5 days;
(3) Replacing the DMEM complete culture solution in the cell culture hole in the step (2) with the cell stain in any one of claims 1 to 3, standing and staining at room temperature for 8-12 min, removing the cell stain, washing the plate with sterile water, observing and indicating cell morphology under an optical microscope after airing, counting cytopathy, and calculating the virus titer.
10. The method for determining the TCID50 of a virus according to claim 9, wherein: the indicator cell is a mouse subcutaneous connective tissue cell, an african green monkey kidney cell, a chinese hamster ovary cell, a mouse embryonic fibroblast, a hamster kidney fibroblast, or a mouse skin fibroblast.
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| CN105300772A (en) * | 2015-09-30 | 2016-02-03 | 成都华西海圻医药科技有限公司 | Wright-Giemsa compound staining solution and preparation method thereof |
| CN105842037A (en) * | 2016-03-21 | 2016-08-10 | 山东农业大学 | Staining method for simultaneously displaying mast cells and acidophilic cells |
| CN112098181A (en) * | 2020-09-09 | 2020-12-18 | 上海皓信生物科技有限公司 | Raji staining solution used based on instrumental method and application thereof |
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| CN105300772A (en) * | 2015-09-30 | 2016-02-03 | 成都华西海圻医药科技有限公司 | Wright-Giemsa compound staining solution and preparation method thereof |
| CN105842037A (en) * | 2016-03-21 | 2016-08-10 | 山东农业大学 | Staining method for simultaneously displaying mast cells and acidophilic cells |
| CN112098181A (en) * | 2020-09-09 | 2020-12-18 | 上海皓信生物科技有限公司 | Raji staining solution used based on instrumental method and application thereof |
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| 任振平: "淋巴细胞微核的两种染色方法检测结果分析", 智慧健康, vol. 4, no. 31, 30 November 2018 (2018-11-30), pages 9 - 10 * |
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