2026-10-13T15:51:19Z https://www.scidb.cn/oai

10.57760/sciencedb.j00217.12451 2026-05-07T11:04:51Z

2026-05-07 Establishment of a Simultaneous Single-Cell Isolation Protocol for Rat Intestinal Tissues and Analysis of Immune Cell Characteristics doi:10.57760/sciencedb.j00217.12451 en Objective: To establish a standardized protocol for the synchronous isolation of high-viability single cells from multiple rat tissues, facilitating flow cytometric analysis of immune cell subsets and functional states across the intestinal tract and associated tissues. Methods: Small intestine, large intestine, mesentery, abdominal fat, Peyer’s patches, and mesenteric lymph nodes were harvested from Sprague-Dawley rats. To prepare single-cell suspensions, intestinal, mesenteric, and adipose tissues underwent sequential chemical and enzymatic digestion, while lymphoid tissues were processed via mechanical dissociation. Results: Single-cell viability exceeded 85% across all samples, with yields ranging from 0.17 to 1.56 × 10⁷ cells per tissue. The distribution of immune cells exhibited distinct tissue-specific patterns:B cells were the predominant population in the majority of analyzed tissues, including Peyer’s patches, mesenteric lymph nodes, and the large intestine. Conversely, the small intestine was characterized by a T-cell-dominant profile, where CD4⁺ T cells accounted for (79.53 ± 7.93)% of the total T-cell population. In vitro stimulation assays revealed that IL-10+ B cells constituted 5.55% of large intestine lymphocytes, and IFN-γ+ T cells accounted for 8.91% of small intestine lymphocytes, demonstrating that this method effectively preserves cellular physiological functions. Compared with enzymatic automatic dissociation, manual dissociation resulted in a 2.9-fold higher cell yield from the large intestine, along with elevated proportions of IL-10+ B cells and IFN-γ+ T cells post-stimulation. Conversely, automatic dissociation yielded marginally higher cell recovery from mesenteric tissues and adipose tissue. Sex had no significant effect on cell yield, viability, or immune cell subset proportions (P>0.05). The method is consistent and robust, making it applicable to both sexes of rats and potentially extendable to other animal models. Conclusion: This standardized, multi-tissue single-cell isolation protocol offers a stable and reproducible framework for flow cytometric analysis, providing a robust methodological foundation for investigating systemic and local immune mechanisms. Multiple Tissues; Single-cell Isolation; Flow Cytometry; Immune Cells CHEN Zhiyu GONG Junwen KE Chaoyun HE Xizi HE Chunye CHEN Yuqing LIU Peiyu Pan Hudan Wang Yiyuan PUBLIC https://creativecommons.org/publicdomain/zero/1.0/ dataset Science Data Bank