Method for mouse peritoneal macrophage phagocytosis experiment and application thereof

Abstract

The invention discloses a mouse peritoneal macrophage phagocytosis experiment method. The method comprises the following steps: step 1, reagent preparation and SPF-level Kunming mouse preparation; 2, macrophage induction: injecting a 6% soluble starch solution into the abdominal cavity of the mouse; 3, inducing phagocytic reaction, namely injecting 1% chicken erythrocyte suspension into the abdominal cavity 48 hours after injecting the 6% soluble starch solution; 4, collecting a sample, injecting the 1% chicken red blood cell suspension for 30 minutes, and taking peritoneal fluid; 5, preparing and dyeing a smear, and clearly distinguishing cell morphology; and 6, carrying out microscopic examination and quantitative analysis, and evaluating the activity of the macrophages by counting the phagocytic percentage and the phagocytic index. The method solves the problems of disordered parameters, difficulty in dyeing identification, extensive results and the like in a traditional method, is simpler and more convenient to operate and higher in result stability, and can provide reliable technical support for immunological research, drug development and disease assessment so as to guarantee the accuracy of experimental data.

Description

Technical Field

[0001] This invention relates to the field of biomedical experimental technology, specifically a standardized method for mouse peritoneal macrophage phagocytosis experiments, applicable to immune function assessment, drug screening, and related disease model research. Background Technology

[0002] Cellular immunity is the core of the body's defense. Macrophages, as key cells of innate immunity, can engulf pathogens and activate specific immunity, playing a crucial role in tumor suppression, immune regulation, and homeostasis maintenance. Precise immune function detection is fundamental to understanding immune mechanisms, helping to reveal the patterns of immune responses in basic research, evaluating the effects of immunomodulators in drug development, and providing a basis for the diagnosis and treatment of immune-related diseases in clinical practice. Current demands for advanced detection technologies require high accuracy, high throughput, and clinical translational capabilities; however, the limitations of existing methods have become a bottleneck for development and urgently need to be overcome.

[0003] Existing methods for detecting immune function include the rosette test and the mouse peritoneal macrophage phagocytosis test. The rosette test uses the binding of E receptors on the surface of T cells with sheep red blood cells to form a rosette, thereby reflecting T cell function. However, it has obvious drawbacks, such as being cumbersome and strict in operation, requiring high aseptic conditions and having a high risk of contamination, requiring strict control of reagents and conditions, being highly temperature-sensitive, having high subjectivity of results, and having a low success rate due to interference from multiple factors.

[0004] The mouse peritoneal macrophage phagocytosis assay assesses macrophage activity by phagocytizing chicken erythrocytes, but it has significant limitations: The lack of standardized parameters is a significant problem: key parameters used to induce macrophage aggregation, such as the concentration of soluble starch (3%-10%) and induction time (24-72 hours), vary considerably across different studies and experimental manuals (see "J Immunol Methods. 1988;112(1):37-42"). This inconsistency directly leads to a coefficient of variation (CV) of over 35% in data from different laboratories, making it difficult to replicate and compare results.

[0005] Staining identification is difficult: The lack of standardization in the selection and operation details of staining methods (such as Giemsa stain and Wright's stain) often leads to unclear cell morphology staining and dark background staining, making it difficult to distinguish macrophages from chicken erythrocytes, resulting in a high misjudgment rate of phagocytic events and poor counting accuracy.

[0006] The analytical methods are crude: most methods only perform qualitative descriptions or randomly select a small number of fields of view (usually ≤3) for simple counting. They lack systematic sampling strategies and statistical analysis, which makes the results highly random and unable to provide accurate quantitative evaluation.

[0007] In summary, the rosette assay is primarily suitable for assessing T cell function in adaptive immunity, but its application in directly assessing macrophage-mediated phagocytic function of innate immunity is limited. Traditional phagocytosis assays, such as the method described in "A Concise Guide to Experimental Cell Biology," typically require the extraction of macrophages via intraperitoneal perfusion, followed by in vitro adhesion, culture, and purification before the phagocytic reaction. This method is not only cumbersome but also leads to unreliable data due to inconsistent parameters (such as varying inducer concentrations and timing). Therefore, there is an urgent need in this field for a macrophage phagocytosis assay method that is standardized in parameters, simple in operation, and provides quantitative results. This method would address the problems of difficult observation of phagocytosis, large counting errors, and poor reproducibility, providing a reliable technical tool for basic immunological research, screening of immunomodulatory drugs, and evaluation of disease models. Summary of the Invention

[0008] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a mouse peritoneal macrophage phagocytosis assay method. This method solves the problems of inconsistent parameters leading to difficulty in observing phagocytosis, large data discrepancies, inaccurate counting due to difficulties in staining identification, and coarse result analysis affecting the reliability of assessment in traditional methods. It simplifies operation, increases the accuracy of results, and standardizes the process. The advantages are highlighted by comparison with the rosette assay, providing efficient and reliable technical support for basic immunological research, drug development, and evaluation of immune disease models.

[0009] The technical solution adopted in this invention is: a method for phagocytosis experiment of mouse peritoneal macrophages, comprising the following steps: Step 1, Pre-experimental preparation: Prepare 6% soluble starch solution, purchase 1% chicken red blood cell suspension and Wright's stain, and select SPF-grade Kunming mice (6-8 weeks old, weighing 18-25g) for acclimatization feeding for 3 days; Step 2, Macrophage Induction: After weighing and fixing the mice, the skin of the lower abdomen was disinfected, and 1 mL of 6% soluble starch solution was injected into the peritoneum. After injection, the abdomen was massaged clockwise for 1 minute, and then the mice were returned to their cages. Step 3, Induction of phagocytic response: 48 hours after starch injection, disinfect the same site and inject 1 mL of 1% chicken red blood cell suspension into the peritoneum (injection rate 0.5 mL / second), massage the abdomen for 30 seconds, and let it stand for 30 minutes; Step 4, Peritoneal fluid collection: 30 minutes after injecting chicken red blood cell suspension, sacrifice the mouse and aspirate the peritoneal fluid (approximately 1-1.5 mL; if the amount is small, add 1 mL of sterile saline to rinse and combine). Step 5, Smear preparation and staining: Take 100 μl of peritoneal fluid, spread it on a slide and air dry it. Add 300 μl of Wright's stain solution and stain for 1-2 minutes. Add an equal volume of PBS buffer (pH=6.4), mix and let stand for 3-5 minutes. Rinse with distilled water until clear and then examine under a microscope. Step 6, Microscopic Examination and Quantitative Analysis: Under oil immersion (1000×), six non-overlapping fields of view were randomly selected, and the total number of macrophages (N) was counted. t ), number of phagocytic positive macrophages (N) p The total number of chicken red blood cells phagocytosed (Nᵣ) was used to calculate the phagocytic percentage (PP = (Nᵣ) / (Nᵣ)). p / N t ()×100%) and phagocytic index (PI=Nᵣ / N) t ).

[0010] In one embodiment, the 6% soluble starch solution needs to be prepared and used immediately, and stored at 4°C. The preparation method is as follows: weigh 6g of soluble starch, add 100mL of sterile physiological saline, stir magnetically until completely dissolved, autoclave at 121°C for 20 minutes, cool to room temperature, and temporarily store in a sealed container at 4°C. It must be used within 24 hours after preparation. The SPF-grade Kunming mice are kept in an environment with a temperature of 22±2°C and a humidity of 50%-60%.

[0011] In one embodiment, during the preparation of the smear, the smear is spread at a uniform speed to avoid cell overlap, and then air-dried at room temperature for about 15 minutes (avoiding direct sunlight); the staining time can be adjusted according to the room temperature, extending it by 1-2 minutes in winter and shortening it in summer.

[0012] In one embodiment, the quantitative analysis requires double-blind counting by two experimenters, and the average value is taken. During the counting, it is necessary to distinguish between intracellular chicken red blood cells and free chicken red blood cells. Macrophages are large, irregular cells with abundant cytoplasm. The criterion for phagocytosis positivity is the presence of ≥1 pale red chicken red blood cell in the cytoplasm.

[0013] The beneficial effects of this invention are as follows: This invention establishes a standardized process by systematically optimizing starch concentration, induction time, staining method, and quantitative standards, significantly reducing the experimental coefficient of variation, improving the accuracy and reproducibility of results, and filling the gap in the precise detection of innate immune function in existing technologies.

[0014] 1. This invention establishes a complete process system by standardizing key parameters (6% soluble starch concentration, 48-hour induction time, Wright staining, and six-field counting method). In particular, for the final functional quantitative indicators (phagocytic percentage and phagocytic index), the intragroup coefficient of variation of the core quantitative indicators of macrophage phagocytic function (phagocytic percentage and phagocytic index) can be reduced from more than 35% in traditional methods to less than 8%, solving the problem of poor reproducibility and achieving accurate and stable assessment of macrophage phagocytic activity.

[0015] 2. The clear staining method and quantitative analysis standard (6 fields of view counting) solve the problems of difficult cell identification and large counting errors, and greatly improve the objectivity and accuracy of the results.

[0016] 3. Compared with the traditional method described in the "Concise Guide to Cell Biology Experiments" which requires in vitro isolation and culture of macrophages, this invention simplifies the operation steps from approximately 12 steps to 6 steps (a reduction of 50%) through in vivo induction and in situ phagocytosis, and shortens the total experimental time from approximately 5.5 hours to 2.5 hours (a reduction of 54.5%). The technical training cycle is shorter, and it can directly assess innate immune function, making it more widely applicable and providing an efficient tool for basic immunological research, drug screening, and disease assessment. Attached Figure Description

[0017] Figure 1 This is a microscopic image (oil immersion, 1000×) of peritoneal fluid cells from the saline control group (48 hours) in Example 1, showing the baseline level of macrophages; Figure 2 The image shows a microscopic examination of peritoneal fluid cells (oil immersion, 1000×) from the 4% soluble starch solution group (48 hours) in Example 1, illustrating macrophage aggregation. Figure 3 The image shows a microscopic examination of peritoneal fluid cells (oil immersion, 1000×) from the 6% soluble starch solution group (48 hours) in Example 1, illustrating macrophage aggregation. Figure 4 The image shows a microscopic examination of peritoneal fluid cells (oil immersion, 1000×) from the 8% soluble starch solution group (48 hours) in Example 1, illustrating macrophage aggregation. Figure 5 This is a microscopic image of peritoneal fluid cells (oil immersion, 1000×) from the group injected 24 hours apart in Example 2, showing phagocytosis. Figure 6 This is a microscopic image of peritoneal fluid cells (oil immersion, 1000×) from the group injected 48 hours apart in Example 2, showing phagocytosis. Figure 7 This is a microscopic image of peritoneal fluid cells (oil immersion, 1000×) from the group injected 72 hours apart in Example 2, showing phagocytosis. Figure 8 This is a microscopic image (oil immersion, 1000×) of peritoneal fluid cells stained with Wright's stain (Solepro) in Example 3, showing phagocytosis. Figure 9 This is a microscopic image (oil immersion, 1000×) of peritoneal fluid cells stained with Wright's stain (Beyotime) in Example 3, showing phagocytosis. Figure 10 This is a microscopic image (oil immersion, 1000×) of peritoneal fluid cells stained with Wright's stain (source leaf) in Example 3, showing phagocytosis. Figure 11This is a microscopic image (oil immersion, 1000×) of peritoneal fluid cells stained with modified Giemsa stain (Beyotime) in Example 3, showing phagocytosis. Figure 12 This is a microscopic image of peritoneal fluid cells stained with Mid-Blue (Reagan) in Example 3 (oil immersion, 1000×), showing phagocytosis. Figure 13 This is a microscopic image of peritoneal fluid cells stained with Wright-Gymsa composite stain (Solepro) in Example 3 (oil immersion, 1000×), showing phagocytosis. Figure 14 This is a microscopic image of peritoneal fluid cells examined using the modified standardized method in Example 4 (oil immersion, 1000×), showing phagocytosis. Figure 15 This is a microscopic image of peritoneal fluid cells from the saline control group in Example 5 (oil immersion, 1000×), showing the phagocytic background level. Figure 16 This is a microscopic image (oil immersion, 1000×) of peritoneal fluid cells from the transfer factor (immunostimulant) treatment group in Example 5, showing enhanced phagocytosis. Figure 17 The image shows a microscopic examination of peritoneal fluid cells (oil immersion, 1000×) from the cyclophosphamide (immunosuppressant) treatment group in Example 5, illustrating phagocytic inhibition. Figure 18 This is a microscopic image of peritoneal fluid from the mouse peritoneal macrophage phagocytosis experiment in Example 6 (Wright stain, oil immersion, 1000×), showing typical phagocytosis phenomena. Figure 19 This is a microscopic image of the rose wreath experiment in Example 6, showing a typical wreath structure and E receptor binding; Figure 20 The image shown is a microscopic examination of the rose wreath experiment in Example 6. The absence of a typical wreath structure indicates that the experiment failed or yielded a negative result. Detailed Implementation

[0018] The present invention will be further described in detail below through examples, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the reagents and materials described are commercially available, and the experimental animals described comply with relevant ethical regulations.

[0019] In all embodiments of this invention, measurement data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, and independent samples t-tests were used for comparisons between two groups. P <0.05 indicates a statistically significant difference.

[0020] Example 1: Screening of soluble starch concentration (based on fixed induction time) Experimental design rationale: To fairly compare the induction effects of different starch concentrations, the effects needed to be evaluated at the same induction time point. 48 hours was selected as a fixed baseline time point to eliminate interference from time variables. The optimal induction concentration was determined by comparing macrophage densities at different concentrations.

[0021] 1. Experimental Design: (1) Grouping and treatment: 50 six-week-old SPF-grade Kunming mice were randomly divided into 10 groups ((3 concentrations × 3 time points) + 1 physiological saline), with 5 mice in each group.

[0022] (2) Concentration factor: 1 mL of soluble starch solution with concentrations of 4%, 6% and 8% (solvent is sterile physiological saline) was injected intraperitoneally. The physiological saline control group was only set at the 48-hour time point (because the baseline value of macrophages in the physiological saline group at 24 hours and 72 hours was extremely low and stable, so there was no need to repeat the setting).

[0023] (3) Time factor: Samples were taken and tested at 24 hours, 48 ​​hours and 72 hours after injection for each group injected with 4%, 6% and 8% soluble starch solution; only the saline control group was set up at the 48-hour time point, and samples were taken and tested at the same time point.

[0024] (4) Detection indicators: cell viability and macrophage density. The detection of macrophage density should be performed according to the following procedure: ① Sample collection: After euthanizing mice at each time point, make a small incision of about 1 cm with ophthalmic scissors 0.5 cm to the left of the abdominal midline (avoiding the intestines and organs), and aspirate peritoneal fluid (about 1-1.5 mL; if the amount is small, add 1 mL of sterile saline to rinse and combine). ② Smear preparation: Take 100 μl of peritoneal fluid, spread it into a smear, and let it air dry (spread the smear at a uniform speed to avoid cell overlap, and let it air dry at room temperature for about 15 minutes, avoiding direct sunlight); ③ Staining: Add 300 μl of Wright's stain solution and stain for 1-2 minutes. Add an equal volume of PBS buffer (pH=6.4), mix, and let stand for 3-5 minutes. Rinse with distilled water until clear. ④ Microscopic counting: Observe under an oil immersion microscope (1000×) and count the number of macrophages to determine their density; at the same time, assess the cell viability, that is, the proportion of surviving cells to the total number of cells.

[0025] (5) Experimental results: Cell survival rate: The survival rate of all starch concentration groups was >90% at each time point, with no difference between groups (P>0.05).

[0026] (6) Macrophage density (×10) 6 The number of cells / mL is shown in Table 1: Table 1 Macrophage density (×10) 6 (pcs / mL)

[0027] 2. Key Conclusions: (1) Time dependence: As can be seen from Table 1, regardless of the concentration of soluble starch injected, the macrophage density reached its peak at 48 hours. The differences between 48 hours and 24 hours and 72 hours in each group were statistically significant (P<0.01).

[0028] (2) Concentration universality: at each independent time point: ①24 hours: The density of the 6% group was significantly higher than that of the 4% and 8% groups (P<0.05).

[0029] ②48 hours: The density in the 6% group was significantly higher than that in the 4% and 8% groups (P<0.01).

[0030] ③ 72 hours: The density in the 6% group was significantly higher than that in the 4% and 8% groups (P<0.05).

[0031] (3) Optimal combination lock: Macrophage density induced by 6% starch + 48 hours (10.6±1.5×10⁻⁶) 6 The concentration-time ratio (p<0.01) was significantly higher than all other concentration-time combinations, making it the optimal parameter.

[0032] In this embodiment, the macrophage density microscopic image (oil immersion, 1000×) of the saline control group (48 hours) is shown below. Figure 1 As shown. Microscopic image of macrophage density in 4% soluble starch solution group (48 hours) (oil immersion, 1000×). Figure 2 As shown. Microscopic image of macrophage density in 6% soluble starch solution group (48 hours) (oil immersion, 1000×). Figure 3 As shown. Microscopic image of macrophage density in 8% soluble starch solution group (48 hours) (oil immersion, 1000×). Figure 4 As shown.

[0033] Example 2: Effect of injection time interval on phagocytic activity (based on optimal starch concentration) Experimental design rationale: After determining the optimal starch concentration (6%), it was necessary to optimize the time interval between starch injection and chicken erythrocyte suspension injection. By setting different time intervals and detecting phagocytic activity indicators, the optimal time point that maximizes macrophage phagocytic capacity was determined.

[0034] 1. Experimental Design: (1) Grouping: Fifteen 6-week-old SPF-grade Kunming mice were randomly divided into 3 groups (n=5).

[0035] (2) Treatment: Mice in all groups were injected intraperitoneally with 1 mL of 6% soluble starch solution.

[0036] (3) Time interval: 1 mL of 1% chicken red blood cell suspension was injected intraperitoneally at 24 hours, 48 ​​hours and 72 hours after the injection of 1 mL of 6% soluble starch solution.

[0037] (4) Detection indicators: 30 minutes after injection of chicken red blood cell suspension, the mice were euthanized by cervical dislocation, peritoneal fluid was aseptically aspirated, and smears were prepared.

[0038] (5) Wright staining: Same as in Example 1.

[0039] (6) Randomly select 6 non-overlapping fields of view under oil immersion and count them: Calculate the percentage of food consumed (PP=(N) p / N t ()×100%) and phagocytic index (PI=Nᵣ / N) t ).

[0040] Where, N t Total number of macrophages (N) t ); N p The number of positive macrophages (phagocytosed ≥1 chicken red blood cell); Nᵣ is the total number of chicken red blood cells phagocytosed.

[0041] 2. Experimental Results: Table 2 Effect of injection time interval on phagocytic activity

[0042] 3. Experimental conclusions: Table 2 shows that, under the condition of induction with 6% soluble starch solution, the phagocytic percentage (PP) and phagocytic index (PI) of the 48-hour time interval group were significantly higher than those of the 24-hour and 72-hour groups (P<0.01). Therefore, 48 hours was determined to be the optimal time interval.

[0043] In this embodiment, the microscopic image (oil immersion, 1000×) of the phagocytic phenomenon results after an injection interval of 24 hours is shown below. Figure 5 As shown. In this embodiment, the microscopic image (oil immersion, 1000×) of the phagocytic phenomenon results after an injection interval of 48 hours is shown. Figure 6 As shown. In this embodiment, the microscopic image (oil immersion, 1000×) of the phagocytic phenomenon results after an injection interval of 72 hours is shown. Figure 7 As shown.

[0044] Example 3: The effect of staining solution type on cell morphology observation Experimental design rationale: Staining results directly affect cell morphology differentiation and counting accuracy. Several commonly used staining solutions were selected and evaluated based on factors such as staining clarity, background interference, and operation time to determine the most suitable staining scheme for quantitative morphological analysis in this experiment.

[0045] 1. Experimental Design: (1) Grouping: 12 smears of peritoneal fluid from the same mouse were prepared, with 2 smears of each staining solution. The staining solutions were Wright's stain (Solebo), Wright's stain (Beyond), Wright's stain (Yuanye), modified Giemsa stain (Beyond), methylene blue stain (Regan), and Wright-Gemsa composite stain (Solebo).

[0046] (2) Evaluation indicators: color clarity, background interference, and operation time.

[0047] (3) Experimental results: Table 3 Comparison of the effects of different staining methods

[0048] Note: Symbol explanation: + quantity indicates the intensity of the effect.

[0049] Conclusion: In terms of morphological observation and statistical analysis of macrophage phagocytosis of chicken erythrocytes, both Wright's stain (Solepro) and Wright's stain (Beyotime) can effectively visualize phagocytosis, meeting the requirements for calculating phagocytosis percentage and phagocytic index. However, Wright's stain (Solepro) has a more significant staining advantage: It allows for clearer differentiation of cell morphology—macrophages appear as deep blue nuclei and pink cytoplasm, while chicken erythrocytes show as pale red ovals, making the procedure more efficient. Therefore, Wright's stain (Solepro) is more suitable for the quantitative morphological analysis in this experiment.

[0050] In this embodiment, the microscopic image (oil immersion, 1000×) of macrophages phagocytizing chicken red blood cells stained with Wright's stain (Solepro) is shown below. Figure 8 As shown. Microscopic image of macrophages phagocytizing chicken erythrocytes stained with Wright's stain (Beyond the Oxygen Plant) (oil immersion, 1000×). Figure 9 As shown. Microscopic image of macrophages phagocytizing chicken erythrocytes stained with Wright's stain (source leaf) (oil immersion, 1000×). Figure 10 As shown. Microscopic image of macrophages phagocytizing chicken erythrocytes stained with modified Giemsa stain (Beyotime) (oil immersion, 1000×). Figure 11 As shown. Microscopic image of macrophages phagocytizing chicken erythrocytes stained with methylene blue (Reagan) solution (oil immersion, 1000×). Figure 12 As shown. Microscopic image of macrophages phagocytizing chicken erythrocytes stained with Wright-Gymsa composite staining solution (Solepro) (oil immersion, 1000×). Figure 13 As shown.

[0051] Example 4: Standardized Method for Modified Mouse Peritoneal Macrophage Phagocytosis Assay Experimental design basis: Integrate the optimal parameters screened in the early stage (6% starch concentration, 48 hours of induction, Wright's stain (Solepro), etc.), standardize reagent preparation, animal treatment, operation procedures and quantitative standards, form a standardized process, and ensure the reproducibility and reliability of the experiment.

[0052] 1. Preparatory work before the experiment (1) Preparation of reagents ①6% soluble starch solution: Weigh 6g of soluble starch, add 100mL of sterile physiological saline, stir magnetically until completely dissolved, autoclave at 121℃ for 20 minutes, cool and store at 4℃ (prepare and use immediately).

[0053] ② Purchase 1% chicken red blood cell suspension and Wright's stain (Solepro).

[0054] ③ Other consumables: disposable 1mL syringes (with 4.5 gauge needles), glass slides (sterilized by wiping with 75% alcohol beforehand), etc.

[0055] Laboratory animal preparation Fifteen 6-week-old SPF-grade Kunming mice were acclimatized for 3 days before the experiment, during which they had free access to food and water. The ambient temperature was 22±2℃ and the humidity was 50%-60%.

[0056] 3. Experimental Procedure (to be performed in stages) (1) Macrophage induction (Day 0) ① After weighing the mice, fix them in place and disinfect the skin on their lower abdomen with 75% alcohol.

[0057] ② Draw 1 mL of 6% soluble starch solution into a 1 mL syringe and insert it into the abdominal cavity at a 45° angle (insertion depth approximately 5 mm, avoiding the bladder and blood vessels). Slowly inject the solution. After injection, gently massage the mouse's abdomen for 1 minute (clockwise) to promote even distribution of the solution, then return it to its cage.

[0058] (2) Phagocytosis response induction (Day 2, 48 hours after starch injection) After repositioning the mouse and disinfecting the same site, inject 1 mL of 1% chicken erythrocyte suspension into the peritoneum at a rate of 0.5 mL / second. Massage the abdomen for 30 seconds to ensure full contact between the chicken erythrocytes and macrophages, then return the mouse to its cage and let it rest for 30 minutes (avoid disturbing the mouse during this period).

[0059] (3) Peritoneal fluid collection (Day 2, 30 minutes after injection of chicken red blood cell suspension) Mice were euthanized by cervical dislocation. The abdominal skin was immediately wiped with 75% alcohol, and the abdominal cavity was cut open (avoiding the intestines and organs). Peritoneal fluid (approximately 1-1.5 mL) was aspirated using a sterile pipette and transferred to a 1.5 mL centrifuge tube. If the amount of peritoneal fluid was small, 1 mL of sterile saline could be injected to flush the peritoneal cavity, and the flushing fluid should be combined (to ensure cell recovery).

[0060] (4) Preparation of smears Take 100 μl of peritoneal fluid and drop it onto one end of a glass slide (1 cm from the edge). Use another glass slide to push the slide at a 45° angle (moving evenly to avoid cell overlap). Allow it to air dry at room temperature (about 15 minutes, avoiding direct sunlight).

[0061] (5) Wright staining Place 300 μl of Wright's stain onto a glass slide and stain for 1-2 minutes to fix the specimen with methanol. Add an equal volume of PBS buffer (pH 6.4), gently agitate the slide to mix the stain with the PBS, and let it stand for 3-5 minutes (until the slide turns purplish-red). Slowly rinse the slide with distilled water (let the water flow down the edge of the slide to avoid direct impact on the cells) until the water becomes clear. Examine the slide under a microscope immediately while still wet, or allow it to dry and then mount it with neutral resin for microscopic examination.

[0062] (6) Microscopic examination and quantitative analysis The counting was conducted by two participants in a double-blind manner, and the observations were performed under an oil immersion microscope (1000×). Six non-overlapping fields of view were randomly selected (avoiding the edges and areas of cell aggregation).

[0063] To minimize subjective bias during the counting process, this experiment employed a double-blind method for microscopic counting, the specific procedures of which are as follows: ① Slide coding: All stained slides are randomly numbered (e.g., using a random number table to generate numbers from 01 to 15) by a third-party experimental staff member (such as a lab administrator or another researcher) who is not involved in the counting process. This staff member records the correspondence between the numbers and the experimental groups (e.g., control group, drug A group, drug B group), and this correspondence table is kept confidential from subsequent counters.

[0064] ② Counting operation: The coded slides are distributed to two experienced counters (counter A and counter B). The counters only know the slide numbers and are completely unaware of the experimental groups and processing information corresponding to the numbers.

[0065] ③ Independent counting: Counters A and B, without knowing each other's results, independently counted the number of macrophages in each of the six fields of view on each slide according to the aforementioned criteria (macrophage detection, phagocytosis positivity detection), and recorded the N value for each slide. t N p N r value.

[0066] ④ Data summarization and decoding: After all slides have been counted, a third party will match and summarize the original data from the two counters with the group correspondence table.

[0067] ⑤ Result Calculation: For each type of slide experimental data, the arithmetic mean of the results from two counters is taken as the final N. t N p N r The values ​​are then used to calculate PP and PI. Additionally, the intragroup correlation coefficient (ICC) or consistency coefficient between the results of the two counters can be calculated to assess the reliability of the data.

[0068] 4. Counting indicators: (1) Total number of macrophages: N t It has the morphology of large, irregular cells with abundant cytoplasm; (2) Number of phagocytic positive macrophages: N p The cytoplasm contains ≥1 pale red chicken erythrocyte; (3) Total number of chicken red blood cells phagocytosed: Nᵣ, it is necessary to distinguish between intracellular chicken red blood cells and free chicken red blood cells; 5. Experimental Calculations (1) Percentage of consuming (PP) = (N p / N t ) × 100% = (33.0 ± 1.8)%; (2) Phagocytic Index (PI) = Nᵣ / N t =2.5±0.6; In this embodiment, the microscopic examination results of the modified mouse peritoneal macrophage phagocytosis phenomenon (oil immersion, 1000×) are as follows: Figure 14 As shown.

[0069] 6. Experimental Conclusions: The improved experimental procedure clearly distinguishes cell morphology, and the quantitative indicators are stable (phagocytosis percentage (33.0±1.8)%, phagocytosis index 2.5±0.6, coefficient of variation <8%), overcoming the shortcomings of traditional methods and providing a reliable standard for macrophage function assessment. This method, through parameter standardization, reduces the experimental coefficient of variation to below 8%, is simple to operate, time-saving, and can directly reflect innate immune function. It is suitable for drug screening and evaluation of immune disease models, providing efficient technical support for immunological research.

[0070] 7. Precautions (1) Avoid cross-contamination between syringe and slide during operation. (2) Pushing the slide too quickly can cause cell rupture, while pushing it too slowly can cause cell accumulation. Practice is required beforehand. (3) The staining time needs to be adjusted according to the room temperature (extend by 1-2 minutes in winter and shorten in summer) to ensure clear contrast between the cell nucleus and cytoplasm. (4) During microscopic examination, two experimenters should count the cells in a double-blind manner and take the average value to reduce errors.

[0071] Example 5: Evaluation of the sustained effects of immunomodulatory drugs based on standardized methods This embodiment aims to verify the application value of the standardized method provided by this invention in evaluating the sustained effects of immunomodulatory drugs. Specifically, by applying drug intervention at the peak of macrophage induction (48 hours after starch injection) and uniformly assessing their phagocytic function 24 hours after drug action (i.e., 72 hours after starch injection), the sustained action of the drug in vivo can be simulated, thereby providing a more comprehensive evaluation of drug efficacy. To accurately separate the drug effect from physiological changes caused by time factors, this experiment established a strictly parallel saline control group. This control group underwent the exact same experimental procedures and time points as the drug-treated group to ensure that the observed differences specifically stemmed from the immunomodulatory effects of the drug.

[0072] 1. Experimental animals: Fifteen 6-week-old SPF-grade Kunming mice were randomly divided into three groups (n=5): immune enhancer group, immunosuppressant group, and saline control group.

[0073] 2. Drug intervention: (1) Immunostimulant group: 1 mL of transfer factor (polypeptide content 5 mg / mL) was injected intraperitoneally 48 hours after starch injection (i.e. Day 2). (2) Immunosuppressant group: 1 mL of cyclophosphamide (concentration 5 mg / mL) was injected intraperitoneally at the same time point; (3) Control group: The same amount of sterile saline was injected into the peritoneum at the same time point.

[0074] 3. Phagocytosis Experiment Procedure: (1) Macrophage induction was performed in each group according to the optimized method: Day 0: 1 mL of 6% starch solution was injected; Drug injection: Day 2: 1 mL of transfer factor / 1 mL of cyclophosphamide / 1 mL of physiological saline was injected; Phagocytosis induction and sample collection: Day 3: 30 minutes after injecting 1 mL of chicken red blood cells, the mice were sacrificed and peritoneal fluid was collected. (2) Prepare smears, stain with Wright's stain (Solebo) and perform microscopic analysis in a unified manner.

[0075] 4. Detection indicator: The total number of macrophages (N) in six non-overlapping fields of view is counted under oil immersion immersion. t ), number of phagocytic positive cells (N) p The total number of chicken red blood cells engulfed (Nᵣ) was used to calculate the phagocytic percentage (PP) and phagocytic index (PI), and statistical methods were used to compare the differences between groups.

[0076] 5. Experimental Results Table 4. Effects of immunomodulatory drugs on macrophage phagocytic activity

[0077] 6. Experimental Conclusions: The results of this experiment showed that, 72 hours after starch injection, the phagocytic activity of the saline control group showed a physiological decline as expected. However, compared with the parallel control group at this specific time point, treatment with the immunostimulant (transfer factor) significantly reversed this decline and increased phagocytic activity to levels far exceeding baseline (P<0.01); while the immunosuppressant (cyclophosphamide) showed a further significant inhibitory effect (P<0.01).

[0078] The above results clearly prove that: (1) The standardized method provided by the present invention can effectively eliminate the interference of confounding factors such as time by setting up strict self-parallel controls, and specifically detect the sustained immunomodulatory effect produced 24 hours after drug treatment.

[0079] (2) This method is not only applicable to assessing the immediate phagocytic function of macrophages, but also a reliable tool for evaluating the sustained potency of immunomodulatory drugs, providing a solid basis for their application in the field of drug screening.

[0080] In this embodiment, the microscopic image (oil immersion, 1000×) of the control (saline) treatment group is as follows: Figure 15 As shown. Microscopic images (oil immersion, 1000×) of the group treated with immune enhancer (transfer factor). Figure 16 As shown. Microscopic images (oil immersion, 1000×) of the immunosuppressant (cyclophosphamide) treated group. Figure 17 As shown.

[0081] Example 6: Multidimensional comparative analysis with the rose wreath experiment (under the same conditions) 1. Comparison of ease of operation Table 5: Comparison of Operational Complexity

[0082] Conclusion: This method omits steps such as density gradient centrifugation and complex temperature control, significantly reducing operational complexity.

[0083] 2. Comprehensive Comparison of Immune Function Reflections This method quantifies the percentage of macrophages phagocytosis of chicken erythrocytes and the phagocytic index, which can directly reflect the phagocytic activity of macrophages, thereby accurately assessing the core function of the body's innate immunity. This provides a direct basis for analyzing the innate immune response mechanism and evaluating the impact of immunomodulators on innate immunity.

[0084] The rosette assay primarily detects the formation of a rosette by binding E receptors on the surface of T cells to sheep erythrocytes. It only reflects the surface receptor status of T cells, and its detection targets are limited to T cells in adaptive immunity. It cannot detect macrophage-mediated innate immune function, and therefore cannot provide a comprehensive assessment of the body's innate immune status. 3. Comparison of Applicable Scope Table 6: Comparison of Application Scenarios

[0085] Comparison of accuracy results (1) Rose wreath experiment procedure ① Reagent and material preparation: Prepare Hank's solution (pH 7.2-7.3), Arne's solution (sterilized and stored), and Giemsa staining working solution (dilute the stock solution with Hank's solution).

[0086] ② Sheep blood processing: Mix sheep venous blood with Alderman's solution at a 1:1 ratio and store at 4°C; before use, wash 3 times with Hank's solution (centrifuge at 500g for 5 minutes) and adjust the concentration to 8-10 times that of T cell suspension.

[0087] ③ T cell suspension preparation: Mouse spleen was ground and sieved to obtain cell suspension; after centrifugation at 400g for 10 minutes, the precipitate was resuspended with sample diluent and the concentration was adjusted to 2×10⁻⁶. 8 -1×10 9 Cells / mL. Take 4 mL of separation solution and 1 mL of cell suspension, centrifuge (400 g, 30 min) to separate the layers, and aspirate the lymphocyte layer; wash and adjust the concentration to 3-5 × 10⁻⁵ cells / mL with Hank's solution. 6 Cells / mL, placed in a 45°C water bath for 30 minutes to remove E receptors.

[0088] ④ Wreath formation and detection: A. Add 0.2 mL of T cell suspension to both the control tube (with 0.1 mL of Hank's solution) and the test tube (with 0.1 mL of transfer factor), and incubate at 37°C for 1 hour.

[0089] B. Add 0.2 mL of SRBC suspension to each tube, shake well, centrifuge at 500 r / min for 3 minutes, and let stand overnight at 4℃.

[0090] C. The next day, aspirate the supernatant, add fixative and let stand for 10 minutes; then add staining solution, stain for 15 minutes and examine under a microscope, count the positive rosettes (lymphocytes bound to ≥3 erythrocytes) and calculate the formation rate.

[0091] ④ Formation rate calculation A. Wreath formation rate: Ten non-overlapping fields of view were randomly selected under oil immersion, and the total number of lymphocytes (N) was counted. t ) and the number of positive wreaths (N)p (i.e., lymphocytes that bind to ≥3 sheep red blood cells), calculation formula: B. Garland formation rate (%) = (N p / N t ) × 100%; C. Repeatability assessment: Double-blind counting by two participants to calculate within-group coefficient of variation (CV) and inter-operator differences: D. Within-group CV = (Standard deviation / Mean) × 100% (CV for this experiment is usually <15%) E. Inter-operator variation = |A count result - B count result| / mean × 100% (usually <20%) (2) Standardized procedure for mouse peritoneal macrophage phagocytosis experiment ① Reagent and material preparation A. Reagents: Prepare 6% soluble starch solution (prepare fresh for immediate use), purchase 1% chicken red blood cell suspension and Wright's stain (Solepro).

[0092] B. Consumables: Disposable 1mL syringe (4.5 gauge needle), glass slides sterilized with 75% alcohol.

[0093] ②Preparation of laboratory animals Fifteen 6-week-old SPF-grade Kunming mice were selected and acclimatized for 3 days. The rearing environment was 22±2℃ and 50%-60% humidity.

[0094] ③ Experimental procedures A. Day 0 (Macrophage Induction): Mice were weighed and fixed, and after disinfection of the lower abdomen, 1 mL of 6% starch solution was injected into the peritoneum. The abdomen was massaged clockwise for 1 minute and then returned to the cage.

[0095] B. Day 2 (Phagocytic response induction): 48 hours later, 1 mL of 1% chicken red blood cell suspension was injected into the same disinfected site (injection rate 0.5 mL / second), massaged for 30 seconds, and left to stand for 30 minutes.

[0096] C. Day 2 (Sample Collection): Mice were sacrificed 30 minutes after injection of chicken red blood cell suspension, and peritoneal fluid (approximately 1-1.5 mL; if the amount is small, physiological saline can be added for flushing) was aspirated.

[0097] D. Smear preparation and staining: Take 100 μl of peritoneal fluid, spread it on a smear and air dry it. Add 300 μl of Wright's stain solution and stain for 1-2 minutes. Add an equal amount of PBS (pH 6.4), mix and let stand for 3-5 minutes. Rinse with distilled water until clear.

[0098] E. Microscopic analysis: Under oil immersion (1000×), six non-overlapping fields of view were randomly selected, and the total number of macrophages (N) was counted. t ), number of phagocytic positive cells (N) p≥1 chicken red blood cell in the cell) and the total number of chicken red blood cells phagocytosed (Nᵣ).

[0099] F. Phagocytosis percentage (PP): Under oil immersion, six non-overlapping fields of view were randomly selected, and the total number of macrophages (N) was counted. t ) and the number of phagocytic positive macrophages (N p (≥1 chicken red blood cell in the cell), calculation formula: PP (%) = (N p / N t ) × 100%; G. Phagocytic Index (PI): Counts the total number of chicken red blood cells engulfed (Nᵣ), calculated using the following formula: PI=Nᵣ / N t ; H. Repeatability assessment: Double-blind counting by two participants, calculation of the coefficient of variation: CV = (Standard deviation / Mean) × 100% (CV < 8% for this experiment) (3) Experimental results: A. Rose wreath experiment: Multiple experiments were conducted and the wreath structure was observed to be few or absent. The judgment was subjective and the wreath formation rate was low. The wreath formation rate (%) was 10 ± 3.1. B. Phagocytosis experiment of mouse peritoneal macrophages: phagocytosis percentage (PP%) = (30.6±2.0), phagocytosis index (PI) = 2.6±0.4.

[0100] (4) Comparison of conclusions: Method accuracy: The mouse peritoneal macrophage phagocytosis experiment measures macrophage function through clear quantitative indicators (PP, PI). The measurement data of phagocytic percentage and phagocytic index have good repeatability and the coefficient of variation (CV) within the group is <8%, indicating high accuracy. In contrast, the rose wreath experiment relies on subjective judgment of wreath formation, and there are large differences between operators, which affects the accuracy of the results.

[0101] In this embodiment, the microscopic images (Wright staining, oil immersion, 1000×) of the mouse peritoneal macrophage phagocytosis experiment are as follows: Figure 18 As shown, from Figure 18 The arrow in the image shows a typical phagocytic phenomenon (as indicated by the arrow in the image). Figure 19 and Figure 20 (Comparison). The results of microscopic examination of rose wreath formation (typical wreath structure) are as follows: Figure 19 As shown, it has a typical wreath structure, such as Figure 19 The image shown by the middle arrow indicates the result of microscopic examination of the rose wreath formation (without wreath structure). Figure 20 As shown.

[0102] The order of each step in the above experimental procedure can be flexibly adjusted according to specific needs in actual operation. At the same time, routine testing can be added, such as quality inspection of the prepared peritoneal fluid smear (to ensure that the cells are evenly distributed and without obvious damage).

[0103] The embodiments described above are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for a mouse peritoneal macrophage phagocytosis experiment, characterized in that, Includes the following steps: Step 1: Experimental preparation: Prepare 6% soluble starch solution, 1% chicken red blood cell suspension and Wright's stain; select 6-8 week old SPF grade Kunming mice weighing 18-25g and acclimatize them for 3 days. Step 2 Macrophage induction: After weighing the mice, disinfect the skin of the lower abdomen with 75% alcohol, inject 1 mL of 6% soluble starch solution into the peritoneum, massage the abdomen clockwise for 1 minute after injection, and return the mice to their cages. Step 3: Phagocytosis response induction: 48 hours after the injection of soluble starch solution, the same site was disinfected and 1 mL of 1% chicken red blood cell suspension was injected into the peritoneum. The abdomen was massaged for 30 seconds and left to stand for 30 minutes. Step 4 Sample collection: 30 minutes after injecting chicken red blood cell suspension, the mice were sacrificed and the peritoneal fluid of the mice was aspirated; Step 5: Smear preparation and staining: Take 100 μl of peritoneal fluid and drop it onto a glass slide, spread the slide and let it dry, add 300 μl of Wright's stain and stain for 1-2 minutes, add an equal volume of PBS buffer (pH=6.4), mix and let stand for 3-5 minutes, then quickly rinse with distilled water to remove excess stain. Step 6: Microscopic Examination and Quantitative Analysis: Under a 1000× oil immersion microscope, six non-overlapping fields of view were randomly selected to count the total number of macrophages (N). t ), number of phagocytic positive macrophages (N) p ) and the total number of chicken red blood cells phagocytosed (Nᵣ), calculate the phagocytic percentage PP=(N p / N t )×100% and the phagocytic index PI=Nᵣ / N t .

2. The method for the mouse peritoneal macrophage phagocytosis experiment according to claim 1, characterized in that, In step 1, the method for preparing the 6% soluble starch solution Includes the following steps: Weigh 6g of soluble starch, add 100mL of sterile physiological saline, stir magnetically until completely dissolved, autoclave at 121℃ for 20 minutes, cool to room temperature, seal and store temporarily at 4℃, and use within 24 hours after preparation.

3. The method for the mouse peritoneal macrophage phagocytosis experiment according to claim 1, characterized in that, In step 1, the ambient temperature for acclimatization feeding for 3 days is 22±2℃ and the humidity is 50%-60%.

4. The method for the mouse peritoneal macrophage phagocytosis experiment according to claim 1, characterized in that, In step 5, the drying process involves air drying at room temperature for 10-15 minutes.

5. The method for the mouse peritoneal macrophage phagocytosis experiment according to claim 1, characterized in that, In step 6, during the counting process, the criteria for determining effective macrophages are: large, irregular cells with abundant cytoplasm; The criteria for identifying phagocytic positive macrophages are: the presence of at least one pale red chicken erythrocyte in the cytoplasm.

6. The method for the mouse peritoneal macrophage phagocytosis experiment according to claim 1, characterized in that, In step 6, a blind counting method is used, and then the average value is taken.

7. The method for the mouse peritoneal macrophage phagocytosis experiment according to claim 1, characterized in that, In step 3, the injection rate of 1% chicken red blood cell suspension into the peritoneum is controlled at 0.5 mL / second.

8. An application of the method as described in any one of claims 1–7 in the screening of immunomodulatory drugs, characterized in that: The regulatory effect of drugs on macrophage phagocytic activity was evaluated by comparing the phagocytic percentage (PP) and phagocytic index (PI) of the drug-treated group with those of the saline control group.

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