A method for determining the value of a standard substance for measuring cell viability by fluorescence labeling and counting

Abstract

The embodiment of the application discloses a kind of standard substance simulation cell viability measurement fixed value method of fluorescent label counting, it is related to biological detection technical field.The method comprises the following steps: preparing fluorescent microsphere suspension;Suspension pretreatment;Drop the suspension in blood cell counting plate counting chamber;Counting under the microscope bright field and dark field environment respectively;According to the counting result of microscope, the luminous efficiency of microsphere, cell viability is calculated;Measure multiple times, take average value as fixed value result;Analysis and calculation of standard substance uncertainty;Verification of fixed value method.The fixed value method has good accuracy, reliability and quantity traceability, and further provides reliable basis for quantitative analysis of cell viability.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a method for determining cell viability using fluorescently labeled counting standard substances. Background Technology

[0002] The detection of biological cell viability plays a crucial role in modern biomedicine. However, the detection of biological cell viability often faces numerous challenges, such as difficulty in detection, difficulty in tracing the source, and difficulties in stability and preservation. Therefore, accurate determination of cell viability is critical. Summary of the Invention

[0003] Therefore, this invention provides a method for simulating cell viability measurement using fluorescently labeled counting standard substances. This invention prepares red and green fluorescent particles to represent dead and live cells, respectively, and uses a microscope for direct counting, providing a new direction for simulating biological cell viability measurement.

[0004] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0005] A method for determining cell viability using fluorescently labeled standard substances, the method comprising:

[0006] (1) Preparation of fluorescent microsphere suspension;

[0007] (2) Suspension pretreatment;

[0008] (3) Add the suspension into the counting chamber of the hemocytometer;

[0009] (4) Count the samples under bright field and dark field conditions of a microscope;

[0010] (5) Calculate the luminescence rate and cell viability of the microspheres based on the microscopic counting results;

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

[0012] (7) Analysis and calculation of the uncertainty of standard materials;

[0013] (8) Verification of the fixed value method.

[0014] Furthermore, the specific details of step (1) are as follows:

[0015] 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;

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

[0017] 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.

[0018] 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.

[0019] Furthermore, the specific details of step (2) are as follows:

[0020] 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.

[0021] Furthermore, the specific details of step (3) are as follows:

[0022] 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.

[0023] Furthermore, the specific details of step (4) are as follows:

[0024] 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.

[0025] Furthermore, in step (5), the formulas for calculating the luminescence rate and cell viability of the microspheres are 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: Suspension concentration, cells / mL;

[0031] N1: Fluorescent particles in a bright-field environment;

[0032] N2: Green fluorescent particles in a dark environment;

[0033] N3: Red fluorescent particles in a dark environment;

[0034] I: Luminescence efficiency of fluorescent microspheres, %

[0035] P: Cell viability, %

[0036] V: Volume of the technical room, mL;

[0037] k: Dilution factor, dimensionless.

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

[0039] Furthermore, the uncertainty introduced by the dilution process includes the uncertainty introduced by the volumetric flask and the uncertainty introduced by the pipette;

[0040] The uncertainties introduced by the microscope measurement process include uncertainties introduced by microscope resolution and uncertainties introduced by measurement repeatability.

[0041] Furthermore, the verification method for the value setting method is performed using a Counstar cell counter.

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

[0043] (1) The present invention ensures that the determination results of the standard substance are highly accurate through a strict preparation process and a precise counting method.

[0044] (2) The present invention utilizes a combination of a hemocytometer and a microscope to improve the repeatability and stability of counting.

[0045] (3) The polystyrene fluorescent microsphere standard material prepared by the method of the present invention has stable chemical properties and luminescence, is stable in storage, and the counting results can be traced back to international or nationally recognized standards, thus ensuring the traceability of the measurement values. Attached Figure Description

[0046] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0047] Figure 1 A flowchart illustrating the method for determining the value of fluorescent microsphere standard material provided by this invention;

[0048] Figure 2 Microscopic images of the fluorescent microspheres provided in Example 1 of the present invention under bright field conditions for particle counting;

[0049] Figure 3 Microscopic images of the fluorescent microspheres provided in Embodiment 1 of the present invention under dark field conditions for particle counting;

[0050] Figure 4 Microscopic images of fluorescent microspheres in bright field environment provided in Example 2 of the present invention;

[0051] Figure 5 This is a microscope image of the fluorescent microspheres provided in Embodiment 2 of the present invention, showing particle counting in a dark field environment. Detailed Implementation

[0052] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] Particle counting was performed using an optical microscope (model BX53, Olympus).

[0054] Example 1: The fluorescent microspheres had a nominal luminescence rate of 100%, a nominal cell viability of 55%, and a nominal total concentration of the fluorescent microsphere standard material of 1.6 × 10⁻⁶. 9 particles / mL

[0055] The particle count standard material consists of monodisperse polystyrene latex particles with an average particle size of 10 μm.

[0056] The above-mentioned method for determining cell viability using fluorescently labeled standard substances includes the following steps:

[0057] (1) 5g of polystyrene microspheres with a particle size of 10μm and 100mL of 0.15% sodium dodecyl sulfonate aqueous solution were added to a three-necked flask. The stirring speed was 350r / min, and the stirring time was 10min at 21℃. 1g of fluorescein 3G and 1g of fluorescein red were ultrasonically mixed with 10mL of 1,2-dichloroethane and added to the above three-necked flask. The reaction was continued for 2h, and the temperature was raised to 68℃ and the reaction was continued for 1h. The solid phase was filtered and repeatedly washed with alcohol and distilled water until it was no longer discolored. Red fluorescent microspheres (dead cells) and green fluorescent microspheres (live cells) were successfully prepared. A dispersion was prepared by mixing sodium dodecylbenzene sulfonate surfactant and distilled water at a mass ratio of 1:99. A certain amount of red fluorescent microsphere and green fluorescent microsphere standard substances were added to a volumetric flask, and the volume was adjusted with the dispersion. The particles were uniformly dispersed in the solution by ultrasound, and a fluorescent microsphere suspension was successfully prepared.

[0058] (2) Take 1 mL of the suspension and transfer it to a 100 mL volumetric flask, and dilute to volume with the dispersion. It is advisable to have 3 to 5 polystyrene fluorescent microspheres in each small square of the hemocytometer. If there are more than 5 polystyrene fluorescent microspheres in each small square, the suspension should be diluted.

[0059] (3) Prepare a clean hemocytometer and coverslip. Place the coverslip on the counting chamber, use a pipette to draw an appropriate amount of suspension, and drop it onto the edge of the coverslip. Let the suspension slowly seep into the counting chamber and fill it all at once to prevent air bubbles from forming. The amount of suspension added should not exceed the rectangular edge between the counting chamber table and the coverslip or coverslip.

[0060] (4) After letting it stand for a moment, place the hemocytometer on the stage and clamp it securely. First, find the counting area under eyepiece magnification ×10 and objective magnification ×10, then switch to eyepiece magnification ×10 and objective magnification ×40 to find the field of view. Count the cells under bright field and dark field conditions of the microscope respectively, and count them directly in one grid.

[0061] (5) The formulas for calculating the luminescence rate and cell viability of the microspheres are as follows:

[0062] C=N1k / V (1)

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

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

[0065] In the formula:

[0066] C: Suspension concentration, cells / mL;

[0067] N1: Fluorescent particles in a bright-field environment;

[0068] N2: Green fluorescent particles in a dark environment;

[0069] N3: Red fluorescent particles in a dark environment;

[0070] I: Luminescence efficiency of fluorescent microspheres, %

[0071] P: Cell viability, %

[0072] V: Volume of the technical room, mL;

[0073] k: Dilution factor, dimensionless.

[0074] (6) Measure multiple times and take the average value as the fixed value result.

[0075] (7) The uncertainty of the standard material is a combination of the uncertainties introduced by the dilution process, the microscopic measurement process, homogeneity, and stability. Among them, the uncertainty introduced by the dilution process includes the uncertainty introduced by the volumetric flask and the pipette, and the uncertainty introduced by the microscopic measurement process includes the uncertainty introduced by the resolution of the microscope and the uncertainty introduced by the measurement repeatability.

[0076] (8) The determination method was verified by using a Counstar cell counter.

[0077] The luminescence efficiency of the fluorescent microspheres was determined to be 99.999%, the cell viability to be 54.98%, and the total concentration of the standard substance in the fluorescent microspheres to be 1.59 × 10⁻⁶. 9 The relative standard deviation of three measurements was 1.19%, and the calculated uncertainty was 3.2%. Validation was performed using a Counstar cell counter (IC1000, USA). The luminescence rate of the fluorescent microspheres was 100%, the cell viability was 54.97%, and the total concentration of the standard substance from the fluorescent microspheres was 1.601 × 10⁻⁶. 9 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.

[0078] Example 2: The fluorescent microspheres had a nominal luminescence rate of 100%, a nominal cell viability of 40%, and a nominal total concentration of 1.0 × 10⁻⁶ for the fluorescent microsphere standard material. 8 particles / mL

[0079] The particle count standard material consists of monodisperse polystyrene latex particles with an average particle size of 10 μm.

[0080] The above-mentioned method for determining cell viability using fluorescently labeled standard substances includes the following steps:

[0081] (1) 4g of 10μm polystyrene microspheres and 150mL of 0.34% sodium dodecyl sulfonate (SDS) were added to a three-necked flask. The stirring speed was 400r / min, and the stirring time was 15min at 25℃. 1.2g of fluorescein 3G and 1.2g of fluorescein red were ultrasonically mixed with 12mL of 1,2-dichloroethane and added to the above three-necked flask. The reaction was continued for 1.5h, and the temperature was raised to 65℃ and the reaction was continued for 1.5h. The solid phase was filtered and repeatedly washed with ethanol and distilled water until it was no longer discolored. Red fluorescent microspheres (dead cells) and green fluorescent microspheres (live cells) were successfully prepared. A dispersion was prepared by mixing sodium dodecylbenzene sulfonate surfactant and distilled water at a mass ratio of 1:99. A certain amount of red fluorescent microsphere and green fluorescent microsphere standard substances were added to a volumetric flask, and the volume was adjusted with the dispersion. The particles were uniformly dispersed in the solution by ultrasound, and a fluorescent microsphere suspension was successfully prepared.

[0082] (2) Take 1 mL of the suspension and transfer it to a 50 mL volumetric flask, and dilute to volume with the dispersion. It is advisable to have 3 to 5 polystyrene fluorescent microspheres in each small square of the hemocytometer. If there are more than 5 polystyrene fluorescent microspheres in each small square, the suspension should be diluted.

[0083] (3) Prepare a clean hemocytometer and coverslip. Place the coverslip on the counting chamber, use a pipette to draw an appropriate amount of suspension, and drop it onto the edge of the coverslip. Let the suspension slowly seep into the counting chamber and fill it all at once to prevent air bubbles from forming. The amount of suspension added should not exceed the rectangular edge between the counting chamber table and the coverslip or coverslip.

[0084] (4) After letting it stand for a moment, place the hemocytometer on the stage and clamp it securely. First, find the counting area under eyepiece magnification ×10 and objective magnification ×10, then switch to eyepiece magnification ×10 and objective magnification ×40 to find the field of view. Count the cells under bright field and dark field conditions of the microscope respectively, and count them directly in one grid.

[0085] (5) The formulas for calculating the luminescence rate and cell viability of the microspheres 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: Suspension concentration, cells / mL;

[0091] N1: Fluorescent particles in a bright-field environment;

[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 materials; (8) Verification of the fixed value method; 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.

2. 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.

3. The method for determining cell viability using fluorescently labeled standard substances as described in 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.

4. 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.

5. The method for determining cell viability using fluorescently labeled standard substances as described in 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.

6. The method for determining cell viability using fluorescently labeled standard substances according to 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.

7. The method for determining cell viability using fluorescently labeled standard substances according to claim 6, 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.

8. 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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