Value fixing method for simulating cell viability measurement by adopting fluorescence labeling counting standard substance

Abstract

The embodiment of the invention discloses a valuing method for simulating cell viability measurement by adopting a fluorescent label counting standard substance, and relates to the technical field of biological detection. The method comprises the following steps: preparing a fluorescent microsphere suspension; pretreating the suspension liquid; dropwise adding the suspension into a counting chamber of a blood counting plate; respectively counting in a bright field environment and a dark field environment of the microscope; calculating the luminous efficiency of the microspheres and the cell viability according to the counting result of the microscope; measuring for multiple times, and taking an average value as a constant value result; analyzing and calculating the uncertainty of the standard substance; and verifying the constant value method. The valuing method provided by the invention has good accuracy, reliability and quantity traceability, and further provides a reliable basis for quantitative analysis of the cell viability.

Description

TECHNICAL FIELD

[0001] The embodiment of the present application relates to the technical field of biological detection, and particularly relates to a kind of fluorescence labeling count standard substance simulation cell activity measurement fixed value method. BACKGROUND

[0002] Biological cell activity detection has a pivotal position in modern biomedical field.However, biological cell activity detection often faces many challenges, such as detection difficulty, traceability difficulty, lack of stability and preservation difficulty and the like problems.Therefore, accurate fixed value of cell activity becomes a key. SUMMARY

[0003] Therefore, the embodiment of the present application provides a kind of fluorescence labeling count standard substance simulation cell activity measurement fixed value method, the present application prepares red fluorescent particles and green fluorescent particles, respectively representing dead cells and living cells, and direct counting is carried out using a microscope, which provides a new direction for simulating biological cell activity measurement.

[0004] In order to achieve the above purpose, the embodiment of the present application provides the following technical scheme:

[0005] A kind of fluorescence labeling standard substance simulation cell activity measurement fixed value method, the method comprises:

[0006] (1) preparing fluorescent microsphere suspension;

[0007] (2) suspension pretreatment;

[0008] (3) drop the suspension in the counting chamber of blood cell counting plate;

[0009] (4) count under the microscope bright field and dark field environment respectively;

[0010] (5) calculate microsphere luminous efficiency and cell activity according to the microscope counting result;

[0011] (6) measure multiple times, and take the average value as the fixed value result;

[0012] (7) analysis and calculation of standard substance uncertainty;

[0013] (8) verification of fixed value method.

[0014] Further, the specific content of step (1) is as follows:

[0015] 4~8g polystyrene microspheres with a particle size of 2~70μm are mixed with 100~500mL 0.1-0.4% sodium dodecyl sulfate aqueous solution, stirred at 20~40℃ for 10~30min, to obtain a first mixture;

[0016] Mix 1-2 g of the dyeing agent with 10-12 mL of 1,2-dichloroethane to obtain a second mixture;

[0017] Mix the first mixture and the second mixture, stir for 1-2 h, then warm to 60-70℃, continue to stir for 1-2 h, filter, and repeatedly wash the obtained solid phase with ethanol and distilled water until no discoloration occurs to obtain red fluorescent microspheres and green fluorescent microspheres;

[0018] Prepare a dispersion liquid with a surfactant mass to distilled water mass ratio of 1:99, take a certain amount of red fluorescent microspheres and green fluorescent microspheres standard substance into a volumetric flask, make up to volume with the dispersion liquid, and uniformly disperse the particles in the solution by ultrasonic to obtain a fluorescent microsphere suspension.

[0019] Further, the specific content of step (2) is as follows:

[0020] It is appropriate that each small square on the hemocytometer contains 3-5 polystyrene fluorescent microspheres, and if each small square contains more than 5 polystyrene fluorescent microspheres, the suspension should be diluted.

[0021] Further, the specific content of step (3) is as follows:

[0022] Prepare a clean hemocytometer and a cover glass, cover the cover glass on the hemocytometer, use a pipette to take an appropriate amount of the suspension, and drop it onto the edge of the cover glass, allowing the suspension to slowly infiltrate the counting chamber, fill the counting chamber at one time, prevent air bubbles, and the amount of the suspension filled is appropriate to not exceed the rectangular edge between the counting chamber table top and the cover or glass.

[0023] Further, the specific content of step (4) is as follows:

[0024] After standing for a while, place the hemocytometer on the stage and clamp it, find the counting area under the low power lens first, then switch to the high power lens to find the field of view, and count under the microscope bright field and dark field environment, respectively, and the counting method is direct grid counting.

[0025] Further, in step (5), the microsphere luminescence rate and cell viability calculation formula is as follows:

[0026] C=N1k / V (1)

[0027] I=(N2+N3) / N1 (2)

[0028] P=N2 / (N2+N3) (3)

[0029] In the formula:

[0030] C: concentration of the suspension, pieces / mL;

[0031] N1: fluorescent particles in bright field environment, number;

[0032] N2: green fluorescent particles in dark field environment, number;

[0033] N3: red fluorescent particles in dark field environment, number;

[0034] I: luminescence rate of fluorescent microspheres, %;

[0035] P: cell viability, %;

[0036] V: volume of technical room, mL;

[0037] k: dilution factor, dimensionless.

[0038] Further, in step (7), the uncertainty of the standard substance includes uncertainty introduced by the dilution process, uncertainty introduced by the microscope measurement process, uncertainty introduced by homogeneity, and uncertainty introduced by stability.

[0039] Further, the uncertainty introduced by the dilution process includes uncertainty introduced by a volumetric flask and uncertainty introduced by a pipette.

[0040] The uncertainty introduced by the microscope measurement process includes uncertainty introduced by microscope resolution and uncertainty introduced by measurement repeatability.

[0041] Further, the verification method of the value assignment method uses a Counstar cell counter.

[0042] The embodiments of the present application have the following advantages:

[0043] (1) The present application ensures that the value assignment result of the standard substance is highly accurate through a strict preparation process and an accurate counting method.

[0044] (2) The present application improves the repeatability and stability of counting by using a combination of a hemocytometer and a microscope.

[0045] (3) The polystyrene fluorescent microsphere standard substance prepared by the method of the present application is chemically stable and luminescent, is stable in storage, and the counting result is traceable to an internationally or nationally recognized standard, thereby ensuring the traceability of the quantity value. BRIEF DESCRIPTION OF DRAWINGS

[0046] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only exemplary, and for those skilled in the art, other drawings can be obtained from the provided drawings without creative labor.

[0047] Figure 1 The value determination method flow chart of the fluorescent microsphere standard substance provided by the present application;

[0048] Figure 2 The microscope photo of the particle counting of the fluorescent microsphere provided by the present application 1 in the bright field environment;

[0049] Figure 3 The microscope photo of the particle counting of the fluorescent microsphere provided by the present application 1 in the dark field environment;

[0050] Figure 4 The microscope photo of the particle counting of the fluorescent microsphere provided by the present application 2 in the bright field environment;

[0051] Figure 5 The microscope photo of the particle counting of the fluorescent microsphere provided by the present application 2 in the dark field environment. DETAILED DESCRIPTION

[0052] The embodiments of the present application will be described in detail by the specific embodiments, and the person skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in the specification. Obviously, the described embodiments are part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without creative labor are within the protection scope of the present application.

[0053] The particle counting is carried out by an optical microscope (model BX53, OLYMPUS).

[0054] The nominal value of the luminous efficiency of the fluorescent microsphere in the embodiment 1 is 100%, the nominal value of the cell viability is 55%, and the nominal value of the total concentration of the fluorescent microsphere standard substance is 1.6×10 9 particles / mL

[0055] The particle counting microparticle standard substance is monodisperse polystyrene latex particles with an average particle size of 10 μm.

[0056] The above-mentioned value determination method for simulating the cell viability measurement by using the fluorescently labeled standard substance includes the following steps:

[0057] (1) Put 5 g polystyrene microspheres with a particle size of 10 μm and 100 mL of 0.15% sodium dodecyl sulfate aqueous solution into a three-necked flask, and stir at a speed of 350 r / min for 10 min at 21 ℃. Mix 1 g of fluorescent yellow 3G and 1 g of fluorescent red with 10 mL of 1,2-dichloroethane uniformly by ultrasonic, and add into the above three-necked flask, and continue to react for 2 h, and then heat to 68 ℃ and react for 1 h. Filter, and wash the obtained solid phase with alcohol and distilled water repeatedly until no discoloration occurs. Red fluorescent microspheres (dead cells) and green fluorescent microspheres (live cells) are successfully prepared. Prepare a dispersion liquid according to a mass ratio of sodium dodecylbenzenesulfonate surfactant to distilled water of 1:99. Take a certain amount of red fluorescent microspheres and green fluorescent microspheres standard substance into a volumetric flask, and make up to the volume with the dispersion liquid. The particles are uniformly dispersed in the solution by ultrasonic, and a fluorescent microsphere suspension is successfully prepared.

[0058] (2) Take 1 mL of the suspension into a 100 mL volumetric flask, and make up to the volume with the dispersion liquid. It is appropriate that 3-5 polystyrene fluorescent microspheres are contained in each small square of the hemocytometer. If more than 5 polystyrene fluorescent microspheres are contained in each small square, the suspension should be diluted.

[0059] (3) Prepare a clean hemocytometer and a cover glass, and cover the cover glass on the hemocytometer. Take an appropriate amount of the suspension with a pipette, and drop it to the edge of the cover glass. Let the suspension slowly infiltrate into the counting chamber, and fill the counting chamber at one time to prevent air bubbles. The amount of the suspension filled is appropriate if it does not exceed the rectangular edge between the counting chamber table top and the cover or the glass.

[0060] (4) Place the hemocytometer on the stage after standing for a while, find the counting area under a magnification of ×10 of the ocular lens and ×10 of the objective lens, and then find the field of view under a magnification of ×10 of the ocular lens and ×40 of the objective lens. Count under the bright field and dark field environments of the microscope respectively, and the counting mode is direct counting by square.

[0061] (5) The microsphere luminescence rate and cell viability calculation formulae are as follows:

[0062] C=N1k / V (1)

[0063] I=(N2+N3) / N1 (2)

[0064] P=N2 / (N2+N3) (3)

[0065] In the formulae:

[0066] C: concentration of the suspension, pieces / mL;

[0067] N1: fluorescent particles in the bright field environment, pieces;

[0068] N2: green fluorescent particles in dark field environment, number of particles;

[0069] N3: red fluorescent particles in dark field environment, number of particles;

[0070] I: luminescence rate of fluorescent microspheres, %;

[0071] P: cell viability, %;

[0072] V: volume of technical room, mL;

[0073] k: dilution factor, dimensionless.

[0074] (6) The average value of multiple measurements is taken as the certified value result.

[0075] (7) The uncertainty of the standard substance is composed of the uncertainties introduced by the dilution process, the microscope measurement process, the uniformity, and the stability. Among them, the uncertainty introduced by the dilution process includes the uncertainties introduced by the volumetric flask and the pipette, and the uncertainty introduced by the microscope measurement process includes the uncertainties introduced by the resolution of the microscope and the measurement repeatability.

[0076] (8) The verification of the certification method is performed by using a Counstar cell counter.

[0077] The certified value result of the luminescence rate of the above fluorescent microspheres is 99.999%, the certified value result of the cell viability is 54.98%, and the certified value result of the total concentration of the fluorescent microsphere standard substance is 1.59×10 9 particles / mL, the relative standard deviation of 3 measurements is 1.19%, and the calculated certified uncertainty is 3.2%. The verification is performed by using a Counstar cell counter (IC1000, USA), the measurement result of the luminescence rate of the fluorescent microspheres is 100%, the measurement result of the cell viability is 54.97%, and the certified value result of the total concentration of the fluorescent microsphere standard substance is 1.601×10 9 particles / mL, which is not much different from the certified value result, indicating that the certification method of the application is accurate, reliable, and practical.

[0078] In Example 2, the nominal value of the luminescence rate of the fluorescent microspheres is 100%, the nominal value of the cell viability is 40%, and the nominal value of the total concentration of the fluorescent microsphere standard substance is 1.0×10 8 particles / mL

[0079] The particle counting microparticle standard substance is monodisperse polystyrene latex particles with an average particle size of 10 μm.

[0080] The above-mentioned certification method for measuring the cell viability by using a fluorescently labeled standard substance includes the following steps:

[0081] (1) 4 g of polystyrene microspheres with a particle size of 10 μm and 150 mL of 0.34% sodium dodecyl sulfate (SDS) were added to a three-necked flask, the stirring speed was 400 r / min, and the stirring time was 15 min at 25°C. 1.2 g of fluorescent yellow 3G and 1.2 g of fluorescent red were uniformly mixed with 12 mL of 1,2-dichloroethane by ultrasonic mixing, and were added to the above three-necked flask, and the reaction was continued for 1.5 h, and the temperature was increased to 65°C, and the reaction was continued for 1.5 h. The obtained solid phase was repeatedly washed with ethanol and distilled water until no discoloration occurred. Red fluorescent microspheres (dead cells) and green fluorescent microspheres (live cells) were successfully prepared. A dispersion liquid was prepared according to a mass ratio of sodium dodecylbenzenesulfonate surfactant to distilled water of 1:99. A certain amount of red fluorescent microspheres and green fluorescent microspheres standard substance was taken into a volumetric flask, and the volume was adjusted with the dispersion liquid. The particles were uniformly dispersed in the solution by ultrasonic, and a fluorescent microsphere suspension was successfully prepared.

[0082] (2) 1 mL of the suspension was taken into a 50 mL volumetric flask, and the volume was adjusted with the dispersion liquid. It is appropriate that 3-5 polystyrene fluorescent microspheres are contained in each small square of the hemocytometer. If more than 5 polystyrene fluorescent microspheres are contained in each small square, the suspension should be diluted.

[0083] (3) A clean hemocytometer and a cover glass were prepared, the cover glass was covered on the hemocytometer, and an appropriate amount of the suspension was taken by a pipette and was dropped to the edge of the cover glass, so that the suspension was slowly infiltrated into the counting chamber. The counting chamber was filled at one time to prevent the generation of air bubbles. The amount of the suspension filled was appropriate to not exceed the rectangular edge between the counting chamber table top and the cover or the glass.

[0084] (4) After standing for a while, the hemocytometer was placed on the stage and was clamped, the counting area was found under a magnification of ×10 of the ocular lens and ×10 of the objective lens, then the ocular lens was converted to a magnification of ×10 and the objective lens was converted to a magnification of ×40 of the high-power lens to find the field of view. The counting was performed under the bright field and the dark field of the microscope, and the counting mode was direct counting by grid.

[0085] (5) The microsphere luminescence rate and the cell viability calculation formula are as follows:

[0086] C = N1k / V (1)

[0087] I = (N2+N3) / N1 (2)

[0088] P = N2 / (N2+N3) (3)

[0089] In the formula,

[0090] C: concentration of the suspension, pieces / mL;

[0091] N1: fluorescent particles in the bright field environment, pieces;

[0092] N2: Green fluorescent particles in a dark environment;

[0093] N3: Red fluorescent particles in a dark environment;

[0094] I: Luminescence efficiency of fluorescent microspheres, %

[0095] P: Cell viability, %

[0096] V: Volume of the technical room, mL;

[0097] k: Dilution factor, dimensionless.

[0098] (6) Measure multiple times and take the average value as the fixed value result.

[0099] (7) The uncertainty of the standard substance is a combination of the uncertainty introduced by the dilution process, the microscopic measurement process, homogeneity, and stability. The uncertainty introduced by the dilution process includes the uncertainty introduced by the volumetric flask and pipette, and the uncertainty introduced by the measurement process includes the uncertainty introduced by the resolution of the microscope and the uncertainty introduced by the measurement repeatability.

[0100] (8) The determination method was verified by using a Counstar cell counter.

[0101] The luminescence efficiency of the fluorescent microspheres was determined to be 99.998%, the cell viability to be 40.01%, and the total concentration of the standard substance in the fluorescent microspheres was determined to be 1.001 × 10⁻⁶. 8 The relative standard deviation of three measurements was 1.17%, and the calculated uncertainty was 3.3%. Validation was performed using a Counstar cell counter (IC1000, USA). The luminescence rate of the fluorescent microspheres was 99.997%, the cell viability was 39.99%, and the total concentration of the standard substance from the fluorescent microspheres was 1.001 × 10⁻⁶. 8 The particle / mL value is not significantly different from the determined value, indicating that the determination method of the present invention is accurate, reliable, and feasible.

[0102] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for determining cell viability using fluorescently labeled standard substances, characterized in that, The method includes: (1) Preparation of fluorescent microsphere suspension; (2) Suspension pretreatment; (3) Add the suspension into the counting chamber of the hemocytometer; (4) Count the samples under bright field and dark field conditions of a microscope; (5) Calculate the luminescence rate and cell viability of the microspheres based on the microscopic counting results; (6) Measure multiple times and take the average value as the fixed value result; (7) Analysis and calculation of the uncertainty of standard substances; (8) Verification of the fixed value method.

2. The method for determining cell viability using fluorescently labeled standard substances as described in claim 1, characterized in that, The specific details of step (1) are as follows: Mix 4-8g of polystyrene microspheres with a particle size of 2-70μm with 100-500mL of 0.1-0.4% sodium dodecyl sulfonate aqueous solution and stir at 20-40℃ for 10-30min to obtain the first mixture; Mix 1-2g of staining agent with 10-12mL of 1,2-dichloroethane to obtain a second mixture; The first mixture and the second mixture were mixed and stirred for 1-2 hours. The temperature was raised to 60-70°C and stirred for another 1-2 hours. The mixture was then filtered. The resulting solid phase was repeatedly washed with ethanol and distilled water until it no longer decolorized, thus obtaining red fluorescent microspheres and green fluorescent microspheres. A dispersion was prepared by mixing surfactant and distilled water at a mass ratio of 1:

99. A certain amount of red fluorescent microspheres and green fluorescent microsphere standard substances were added to a volumetric flask, and the volume was adjusted with the dispersion. The particles were then uniformly dispersed in the solution by ultrasound to obtain a fluorescent microsphere suspension.

3. The method for determining cell viability using fluorescently labeled standard substances as described in claim 1, characterized in that, Step (2) details are as follows: Ideally, each small square on the hemocytometer should contain 3 to 5 polystyrene fluorescent microspheres. If each small square contains more than 5 polystyrene fluorescent microspheres, the suspension should be diluted.

4. The method for determining cell viability using fluorescently labeled standard substances according to claim 1, characterized in that, Step (3) details are as follows: Prepare a clean hemocytometer and coverslip. Place the coverslip on the counting chamber, use a pipette to draw an appropriate amount of suspension, and add it to the edge of the coverslip. Allow the suspension to slowly seep into the counting chamber and fill it completely at once to prevent air bubbles from forming. The amount of suspension added should not exceed the rectangular edge between the counting chamber platform and the coverslip or coverslip.

5. The method for determining cell viability using fluorescently labeled standard substances as described in claim 1, characterized in that, Step (4) details are as follows: After letting it stand for a moment, place the hemocytometer on the stage and clamp it securely. First, locate the counting area under low magnification, then switch to high magnification to locate the field of view. Count the cells under both bright and dark field conditions of the microscope, using direct, segment-by-segment counting.

6. The method for determining cell viability using fluorescently labeled standard substances according to claim 1, characterized in that, In step (5), the formulas for calculating the luminescence rate and cell viability of the microspheres are as follows: C=N1k / V (1) I=(N2+N3) / N1 (2; P=N2 / (N2+N3) (3; In the formula: C: Suspension concentration, cells / mL; N1: Fluorescent particles in a bright-field environment; N2: Green fluorescent particles in a dark environment; N3: Red fluorescent particles in a dark environment; I: Luminescence efficiency of fluorescent microspheres, % P: Cell viability, % V: Volume of the technical room, mL; k: Dilution factor, dimensionless.

7. The method for determining cell viability using fluorescently labeled standard substances as described in claim 1, characterized in that, In step (7), the uncertainty of the standard substance includes the uncertainty introduced by the dilution process, the uncertainty introduced by the microscopic measurement process, the uncertainty introduced by the homogeneity, and the uncertainty introduced by the stability.

8. The method for determining cell viability using fluorescently labeled standard substances according to claim 7, characterized in that, The uncertainty introduced by the dilution process includes the uncertainty introduced by the volumetric flask and the uncertainty introduced by the pipette; The uncertainties introduced by the microscope measurement process include uncertainties introduced by microscope resolution and uncertainties introduced by measurement repeatability.

9. The method for determining cell viability using fluorescently labeled standard substances according to claim 1, characterized in that, The verification method for the value determination method was performed using a Counstar cell counter.

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