High activity of intestinal lamina propria immune cells in chicken and separation method by multicolor flow cytometry analysis

Abstract

This invention belongs to the field of animal immunology technology, specifically disclosing a method for highly active chicken intestinal lamina propria immune cells and their separation by multicolor flow cytometry, including: tissue pretreatment and predigestion, enzymatic digestion and dissociation, density gradient centrifugation enrichment, cell recovery, and identification of chicken intestinal lamina propria immune cells. The single-cell suspension prepared by this invention has few impurities and a clear background, and contains high levels of helper T cells (CD4+). + CD8 ‑ The proportion of cytotoxic T cells (CD8+) increased from 18.8% to 36.09%, and the proportion of CD8+ T cells increased. + CD4 ‑ The proportion of [unclear - possibly related to immune cell composition] increased from 1.15% to 10.85%, and this ratio more accurately reflects the composition of immune cells in the intestinal lamina propria; the false-positive expression rate of activation markers such as TCR and CD25 or pan-T markers decreased from 41.07% to 1.83%, while the proportion of B cells (Bu-1) increased. + ) and macrophages (M0 + The detection ratio is also more reasonable, thereby ensuring the accuracy of streaming clustering and the reliability of data.

Description

Technical Field

[0001] This invention relates to the field of animal immunology technology, and in particular to a method for the separation of highly active and multicolor flow cytometry-based immune cells in the lamina propria of the chicken intestine. Background Technology

[0002] The lamina propria of the chicken intestine is an important component of the digestive tract mucosa, located below the epithelial layer. It is mainly composed of connective tissue and is a key area for intestinal immune defense and nutrient absorption.

[0003] Currently, the commonly used method for separating chicken intestinal immune cells is the grinding separation method, which involves physical grinding or a combination of chemical digestion and physical grinding. However, the lamina propria of the chicken intestine has a dense structure, rich in collagen and reticular fibers, and avian immune cells differ significantly from mammalian immune cells in terms of physical toughness and enzyme sensitivity. Traditional physical grinding methods or common enzymatic digestion methods (such as using collagenase IV alone) generally present a contradiction when processing chicken intestinal tissue: incomplete cell dissociation and severe damage to cell viability. Over-digestion or grinding leads to the death of a large number of lymphocytes (especially vulnerable T cell subsets); while insufficient digestion fails to effectively release immune cells from the deep lamina propria, resulting in low yield and the presence of a large number of epithelial cells and dead cell fragments.

[0004] More importantly, the cell suspension obtained by the above methods severely interferes with the accurate analysis of subsequent flow cytometry. Low cell viability directly leads to enhanced non-specific staining and increased fluorescence background; while excessive cell debris and contaminating cells can clog the flow cytometer's sample inlet tube and cause severe fluorescence signal interference, making it difficult to detect key immune subsets (such as CD4). + T, CD8 + T cells are difficult to accurately delineate and quantify, resulting in a high false positive rate and failing to truly reflect the intestinal immune status.

[0005] Therefore, developing a separation method that can efficiently dissect the lamina propria of the chicken intestine while maximally preserving the integrity and activity of immune cells, and is suitable for high-resolution multicolor flow cytometry analysis, is an urgent need and a technical challenge in poultry mucosal immunology research. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for separating highly active immune cells from the lamina propria of the chicken intestine using multicolor flow cytometry.

[0007] To achieve the above objectives, the present invention is implemented according to the following technical solution: A highly active and multicolor flow cytometry-based method for separating immune cells from the lamina propria of the chicken intestine includes the following steps: S1. Tissue pretreatment and predigestion: Chicken intestinal tissue is taken, cleaned, and shredded, and then shaken in a predigestion solution to remove epithelial cells and intraepithelial lymphocytes; the predigestion solution is a balanced salt solution containing EDTA, dithiothreitol (DTT) and fetal bovine serum. S2, Enzymatic digestion and dissociation: The pre-digested tissue fragments are transferred to a digestion solution for shaking digestion; the digestion solution is RPMI 1640 medium containing collagenase D, collagenase VIII and deoxyribonuclease DNase I; S3. Density gradient centrifugation enrichment: Collect cells by centrifugation of the digested single-cell suspension, resuspend the cell pellet in Percoll solution with a volume concentration of 35-45%, and stack Percoll solution with a volume concentration of 70-85% at the bottom to form a discontinuous density gradient for centrifugation. S4. Cell recovery: Collect the cell pellet located between the two Percoll solution interfaces, and resuspend the cell pellet with PBS buffer to obtain a suspension of chicken intestinal lamina propria immune cells. S5. Identification of chicken intestinal lamina propria immune cells: Using the obtained chicken intestinal lamina propria immune cell suspension, the expression of CD3, CD4, CD8, CD25, TCR, Bu-1 and M0 molecules were simultaneously detected by multicolor flow cytometry to distinguish and quantify T cell subsets, B cells and macrophages.

[0008] Furthermore, the concentration of EDTA in the pre-digested solution is 5-10 mM, the concentration of dithiothreitol (DTT) is 0.5-2 mM, and the volume percentage of fetal bovine serum is 5-10%; the balanced salt solution is an HBSS balanced salt solution.

[0009] Furthermore, in step S1, the mixture is subjected to oscillation treatment at 35-38 ℃ and 200-250 rpm for 15-25 min.

[0010] Furthermore, the final concentration of collagenase D in the digestive fluid is 80-120 U / mL, the final concentration of collagenase VIII is 80-120 U / mL, and the final concentration of deoxyribonuclease DNase I is 5-15 U / mL.

[0011] Furthermore, in step S2, the digestion is carried out by shaking at 35-38 ℃ and 200-250 rpm for 25-35 min.

[0012] Furthermore, in step S3, centrifugation is performed for 15-25 minutes under a relative centrifugal force of 800-1200 g.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1) This invention, through an optimized two-step enzymatic digestion system (a specific concentration of collagenase D / VIII combined with DNase I), gentle oscillation conditions, and precise selection of the subsequent density gradient centrifugation medium, significantly reduces the mortality rate of isolated immune cells from 37.01% in the traditional grinding method to 13.66%, and significantly improves cell viability, providing high-quality live cell samples for subsequent long-term experiments.

[0014] 2) The single-cell suspension prepared by this invention has few impurities and a clear background, and contains helper T cells (CD4+). + CD8 - The proportion of cytotoxic T cells (CD8+) increased from 18.8% to 36.09%, and the proportion of CD8+ T cells increased. + CD4 - The proportion of TCR and CD25 activation or pan-T marker expression increased from 1.15% to 10.85%, and this ratio more accurately reflects the composition of immune cells in the intestinal lamina propria; the false-positive expression rate of TCR, CD25, and other activation or pan-T markers decreased from 41.07% to 1.83%, while the proportion of B cells (Bu-1) increased. + ) and macrophages (M0 + The detection ratio is also more reasonable, thereby ensuring the accuracy of streaming clustering and the reliability of data.

[0015] 3) The enzymatic hydrolysis and separation scheme customized for the characteristics of chicken intestinal tissue in this invention overcomes the influence of individual differences and batch differences in tissue, and provides a stable and reliable technical platform for quantitative research on chicken intestinal mucosal immunity, vaccine potency evaluation and disease model analysis. Attached Figure Description

[0016] Figure 1 A schematic diagram of the effect of density gradient centrifugation on immune cells in the lamina propria of the chicken intestine: (a) the cell suspension state before centrifugation (cells were resuspended in 40% Percoll solution and then 80% Percoll solution was added to the bottom); (b) the typical cell layering interface formed after centrifugation.

[0017] Figure 2 The diagrams show flow cytometry gating strategies for total immune cells in the lamina propria of the chicken intestine obtained by different isolation methods; (a) is a gating strategy diagram for T cell lines; (b) is a gating strategy diagram for B cells, monocytes, and macrophages.

[0018] Figure 3 The following is a statistical comparison of the live / dead cell (Live / Dead staining) viability of chicken intestinal lamina propria immune cells obtained by different isolation methods: (a) is a graph showing the proportion of dead cells in chicken intestinal lamina propria immune cells obtained by method one; (b) is a graph showing the proportion of dead cells in chicken intestinal lamina propria immune cells obtained by method two; and (c) is a graph showing the proportion of dead cells.

[0019] Figure 4 CD45 in chicken intestinal lamina propria immune cells obtained using different isolation methods + Leukocyte analysis results: (a) CD45 in chicken intestinal lamina propria immune cells obtained by method one. + (a) White blood cell scatter plot; (b) CD45 in chicken intestinal lamina propria immune cells obtained by method two. + (c) is a scatter plot of white blood cells; (d) is a statistical plot of cell proportions.

[0020] Figure 5 CD3+ in chicken intestinal lamina propria immune cells obtained using different isolation methods + Leukocyte analysis results: (a) shows CD3 in chicken intestinal lamina propria immune cells obtained by method one. + (a) White blood cell scatter plot; (b) CD3 in chicken intestinal lamina propria immune cells obtained by method two. + (c) is a scatter plot of white blood cells; (d) is a statistical plot of cell proportions.

[0021] Figure 6 CD4+ in chicken intestinal lamina propria immune cells obtained using different isolation methods + CD8 - Helper T cell analysis results: (a) CD4 cells in chicken intestinal lamina propria immune cells obtained by method one. + CD8 - (a) Scatter plot of helper T cells; (b) CD8 cells in chicken intestinal lamina propria immune cells obtained by method two. + CD4 - Scatter plot of helper T cells; (c) shows CD4. + CD8 - Statistical chart of helper T cell proportions; (d) represents CD8. + CD4 - Statistical chart of helper T cell proportions.

[0022] Figure 7 CD25 in chicken intestinal lamina propria immune cells obtained using different isolation methods + Cell analysis results: (a) CD25 in chicken intestinal lamina propria immune cells obtained by method one. + (a) Cell scatter plot; (b) CD25 in chicken intestinal lamina propria immune cells obtained by method two. + (c) Cell scatter plot; (d) Cell proportion statistics.

[0023] Figure 8 TCR in chicken intestinal lamina propria immune cells obtained using different isolation methods + Cell analysis results: (a) TCR in chicken intestinal lamina propria immune cells obtained by method one+ (a) Cell scatter plot; (b) TCR in chicken intestinal lamina propria immune cells obtained by method two + (c) Cell scatter plot; (d) Cell proportion statistics.

[0024] Figure 9 Bu-1 in chicken intestinal lamina propria immune cells obtained using different isolation methods + B cell analysis results: (a) Bu-1 in chicken intestinal lamina propria immune cells obtained by method one. + (b) Scatter plot of B cells; (b) Bu-1 in chicken intestinal lamina propria immune cells obtained by method two. + (b) is a scatter plot of cells; (c) is a statistical chart of cell proportions.

[0025] Figure 10 To identify M0 in chicken intestinal lamina propria immune cells obtained using different isolation methods + Macrophage analysis results: (a) shows the M0 in chicken intestinal lamina propria immune cells obtained by method one. + (a) Scatter plot of macrophages; (b) M0 in chicken intestinal lamina propria immune cells obtained by method two. + (c) Scatter plot of macrophages; (d) is a statistical plot of cell proportions. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0027] Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available.

[0028] To obtain lamina propria immune cells from chicken intestines, 12 one-day-old yellow-feathered broilers were pre-raised for one week in a standardized chicken house with identical rearing conditions. They were fed a basal diet, and a completely randomized block design was used, randomly dividing the chicks into two treatment groups with six replicates per group. One group was used for preparing lamina propria immune cells by grinding, and the other group was used for preparing lamina propria immune cells by enzymatic digestion and gradient separation. All chicks were housed in single-layer cages with free access to feed and water, and under a continuous lighting regime.

[0029] After feeding for one week, chicken intestinal tissue was taken, including 4 cm of ileum (representing small intestine tissue) and colon (representing large intestine tissue). The adipose tissue was removed, the intestinal tissue was longitudinally dissected, and the tissue was placed in a culture dish of pre-cooled 1×PBS for washing. After cleaning, the intestine was cut into 0.5-1 cm segments.

[0030] Comparative Example 1: Preparation of chicken intestinal lamina propria immune cells using conventional grinding method (referred to as Method 1) The shredded intestinal tissue fragments were immersed in 5 mL of pre-digestion solution (5 mL 1×PBS and 50 μL 0.5 M EDTA). 5 μL of 1 m M DTT was added to each tube, and the tubes were placed upright in a benchtop shaker at 37 °C and 200 rpm for 20 min of horizontal shaking. After pre-digestion, the solution was discarded, and the tissue fragments were collected. The tissue fragments were washed once with 1×PBS to remove EDTA, and then 1 mL of 1×PBS and 3-4 grinding beads were added. The tubes were then ground at 40 Hz for 20 s. After grinding, 1 mL of digestion solution (0.5 μL collagenase D + 0.5 μL DNase I + 1 mL 1×PBS) was added to each tube. The tubes were shaken at 37 °C and 200 rpm for 20 min. After digestion, the tissue fragments were filtered through a 70 μm filter, and the filtrate was collected. If connective tissue was still visible in the filter, the digestion steps were repeated with the addition of an appropriate amount of digestion solution. The filtrate was centrifuged at 500×g for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL of 1×PBS to obtain a chicken intestinal lamina propria immune cell suspension. Viable cell counting was then performed to ensure that each tube contained 1×102 cells. 6 Add the required liquid volume, centrifuge again at 500×g for 5 min, discard the supernatant, and resuspend in 200 μL of 1×PBS for subsequent flow cytometry operations.

[0031] Example 1: Preparation of chicken intestinal lamina propria immune cells by enzymatic digestion and gradient separation according to the present invention (referred to as Method 2). The shredded intestinal tissue fragments were immersed in a 15 mL centrifuge tube containing 10 mL of pre-digested intestinal tissue. The tube was then placed upright on a benchtop shaker at 37 ℃ and 250 rpm for 20 min, followed by rapid up-and-down shaking for 1 min. The fragments were then washed three times with pre-cooled 1×PBS to remove residual EDTA. After washing, the intestinal segments were transferred to a 15 mL centrifuge tube containing 3 mL of digestion solution and placed upright on a shaker at 37 ℃ and 250 rpm for 30 min, followed by rapid up-and-down shaking for 1 min. If the chickens were older and a larger amount of intestinal tissue was collected, this step could be repeated once. The digested tissue fragments, along with the digestion solution, were then pipetted through a 70 μm filter membrane into a 50 mL centrifuge tube. The volume was adjusted to 10 mL with 1×PBS, and the tube was centrifuged at 800×g at room temperature for 5-10 min. The supernatant was discarded.

[0032] Resuspend the cell pellet in 4 mL of 40% Percoll solution, and add 2.5 mL of 80% Percoll solution to the bottom of the centrifuge tube using a dropper (see [link to centrifuge tube]). Figure 1 (a) Centrifuge at 1000×g at room temperature for 20 min (5 increments, 1 decrement). After centrifugation, the presence of distinct layers indicates successful separation (see [reference]). Figure 1(b) First, remove the upper layer of impurities, then carefully aspirate the cell pellet from the middle layer using a dropper, transfer it to a 15 mL centrifuge tube, bring the volume to 14 mL with PBS buffer, centrifuge at 800×g at room temperature for 10 min, and discard the supernatant. Resuspend the cell pellet in 1 mL of 1×PBS containing 2% FBS to obtain a suspension of chicken intestinal lamina propria immune cells. Take 10 μL of the separated single-cell suspension and perform cell counting to calculate the number of cells per tube to be 1×10⁻⁶. 6 The required liquid volume was centrifuged again at 500×g at room temperature for 5 min. The supernatant was discarded, and the mixture was resuspended in 200 μL of 1×PBS containing 2% FBS for subsequent flow cytometry operations.

[0033] After separating chicken intestinal lamina propria immune cells using the two methods described above, the corresponding fluorescently labeled antibodies (Live / Dead, CD45, CD3, CD4, CD25, CD8a, TCR, M0, Bu-1) were added according to the manufacturer's instructions, and the cells were incubated at 4°C in the dark for 30 min. The cells were then centrifuged at 500×g for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL of PBS. Flow cytometry data were analyzed using FlowJo. For specific gating strategies, please refer to [link to relevant documentation]. Figure 2 Specific test results are as follows: Figures 3-9 As shown.

[0034] like Figure 3 As shown, the proportion of dead immune cells obtained by method 1 was 37.01%, while the proportion of dead immune cells obtained by method 2 was 13.66%. The proportion of live cells obtained by method 2 was significantly higher than that of method 1, indicating that the method of the present invention has excellent activity protection effect on immune cells of the lamina propria of the chicken intestine.

[0035] like Figure 4 As shown, the white blood cells (CD45) obtained by method one are... + The proportion of method 1 is 36.61%, while that of method 2 is 24.17%.

[0036] like Figure 5 As shown, total T cells (CD3+) obtained by method one + The proportion of method 1 is 51.61%, while that of method 2 is 29.34%.

[0037] like Figure 6 As shown, the helper T cell subset (CD4) obtained by method one + CD8 - in CD3 + The proportion of cytotoxic T cell subsets obtained by method one was 18.8%, significantly lower than the 36.09% obtained by method two; + CD4 - in CD3+ The proportion was 1.15%, significantly lower than the 10.85% of Method 2.

[0038] like Figure 7 As shown, CD25 obtained by separating using Method 1 + Cells (CD25) + in CD3 + The proportion of method 1 is 41.07%, while that of method 2 is 1.83%.

[0039] like Figure 8 As shown, TCR cells (TCR) obtained by method one + in CD3 + The proportion of method 1 is 73.4%, while that of method 2 is 9.32%.

[0040] like Figure 9 As shown, the B lymphocytes (Bu-1) obtained by method one + The proportion of method 1 is 60.19%, while that of method 2 is 21.73%.

[0041] like Figure 10 As shown, macrophages (M0) obtained by method one + in CD45 + The proportion of method 1 is 54%, while that of method 2 is 28.80%.

[0042] In summary, this invention, by comparing the effects of two separation methods on immune cells in the lamina propria of the chicken intestine, found that Method 2 is superior to Method 1 in both cell viability protection and sorting accuracy. Specifically, Method 2 significantly reduced the proportion of dead cells, indicating a stronger protective effect on cell viability. In the analysis of immune cell subsets, although Method 2 resulted in a decrease in total white blood cells (CD45... + ) and total T cells (CD3) + The proportion of CD4+ cells was low, but the number of helper T cells (CD4+) was significantly increased. + CD8 - ) and cytotoxic T cells (CD8) + CD4 - The proportion of TCR was [not specified]. Meanwhile, Method 2 significantly reduced the TCR [not specified]. + CD25 + B cells (Bu-1) + ) and macrophages (M0 +The non-specific expression of markers such as ) reflects its ability to effectively reduce false positives in staining and improve the accuracy of clustering. The principle of this invention is as follows: Chicken intestinal lamina propria is rich in connective tissue. A complex enzyme system of collagenase D and collagenase VIII is used to hydrolyze type I and type VIII collagen more efficiently and gently, avoiding cell damage caused by using a single potent protease. The addition of DNase I effectively degrades DNA released due to cell damage, reducing cell clumping and viscosity during flow cytometry. The pre-digestion step of 5 mM EDTA and 1 mM DTT is crucial for chicken intestinal tissue, effectively chelating calcium ions and opening disulfide bonds. This loosens epithelial connections without the use of potent proteases, laying the foundation for subsequent gentle enzymatic digestion. This step is key to protecting cell viability. The 40% / 80% Percoll discontinuous density gradient is the result of extensive pre-experiment optimization. This density range most effectively separates active lymphocytes, monocytes / macrophages from dead cells, debris, and most epithelial cells in the chicken intestinal lamina propria. This specific ratio is not a conventional or known choice in the art.

[0043] In summary, the method of this invention not only optimizes cell survival rate but also enhances the reliability of immune cell sorting, providing more efficient and accurate technical support for related research.

[0044] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for highly active and multicolor flow cytometry-based separation of immune cells in the lamina propria of the chicken intestine, characterized in that, Includes the following steps: S1. Tissue pretreatment and predigestion: Chicken intestinal tissue is taken, cleaned, and shredded, and then shaken in a predigestion solution to remove epithelial cells and intraepithelial lymphocytes; the predigestion solution is a balanced salt solution containing EDTA, dithiothreitol (DTT) and fetal bovine serum. S2, Enzymatic digestion and dissociation: The pre-digested tissue fragments are transferred to a digestion solution for shaking digestion; the digestion solution is RPMI 1640 medium containing collagenase D, collagenase VIII and deoxyribonuclease DNase I; S3. Density gradient centrifugation enrichment: Collect cells by centrifugation of the digested single-cell suspension, resuspend the cell pellet in Percoll solution with a volume concentration of 35-45%, and stack Percoll solution with a volume concentration of 70-85% at the bottom to form a discontinuous density gradient for centrifugation. S4. Cell recovery: Collect the cell pellet located between the two Percoll solution interfaces, and resuspend the cell pellet with PBS buffer to obtain a suspension of chicken intestinal lamina propria immune cells. S5. Identification of chicken intestinal lamina propria immune cells: Using the obtained chicken intestinal lamina propria immune cell suspension, the expression of CD3, CD4, CD8, CD25, TCR, Bu-1 and M0 molecules were simultaneously detected by multicolor flow cytometry to distinguish and quantify T cell subsets, B cells and macrophages.

2. The method for high-activity and multicolor flow cytometry separation of chicken intestinal lamina propria immune cells according to claim 1, characterized in that, The pre-digested solution contains 5-10 mM EDTA, 0.5-2 mM dithiothreitol (DTT), and 5-10% fetal bovine serum by volume; the balanced salt solution is an HBSS balanced salt solution.

3. The method for high-activity and multicolor flow cytometry separation of chicken intestinal lamina propria immune cells according to claim 1, characterized in that, In step S1, the mixture is subjected to oscillation treatment at 35-38 ℃ and 200-250 rpm for 15-25 min.

4. The method for high-activity and multicolor flow cytometry separation of chicken intestinal lamina propria immune cells according to claim 1, characterized in that, The final concentration of collagenase D in the digestive fluid is 80-120 U / mL, the final concentration of collagenase VIII is 80-120 U / mL, and the final concentration of deoxyribonuclease DNase I is 5-15 U / mL.

5. The method for highly active and multicolor flow cytometry-based separation of chicken intestinal lamina propria immune cells according to claim 1, characterized in that, In step S2, the food is digested by shaking at 35-38 ℃ and 200-250 rpm for 25-35 minutes.

6. The method for highly active and multicolor flow cytometry-based separation of chicken intestinal lamina propria immune cells according to claim 1, characterized in that, In step S3, centrifugation is performed for 15-25 minutes under a relative centrifugal force of 800-1200 g.

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