Comparison of methods for the preparation of single-cell suspensions of rat retina and immunophenotyping by flow cytometry

IF 2.7 4区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Neuroscience Methods Pub Date : 2025-02-07 DOI:10.1016/j.jneumeth.2025.110384

Qingyao Wang , Xiandan Zhu , Xing Zhang , Yong Xia , Xuesong Tian

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引用次数: 0

Abstract

Background

Although the eye has traditionally been considered an immune-privileged organ, progressive studies have re-evaluated the role of the immune response in retinopathy. The application of flow cytometry for immunophenotyping analysis of retinal single-cell suspensions has gradually attracted attention.

New methods

We dissociated the retinal tissue using trypsin digestion, papain digestion, mechanical grinding treatment and liberase + DNase Ⅰ digestion, respectively. Subsequently we assessed the quality of cell suspension (clumping rate, concentration and viability) with two different dyes, Trypan Blue (TPB) and Acridine Orange/Propidium Iodide (AO/PI). We distinguished T cells and their sub-populations by flow cytometry. Furthermore, we analyzed their immune responses to evaluate those four methods for preparing retinal single-cell suspension.

Results

The AO/PI staining method enables a rapid and precise evaluation of cell quality of retinal single cell suspensions, while TPB staining has limitations. Flow cytometric analysis showed that single cell suspensions dispersed with papain and trypsin exhibited reduced cell adhesion. However, trypsin digestion may affect antibody binding. The mechanical grinding treatment reduced cell yield and was prone to double-positivity. The number of cells within the cell-circle gate is significantly limited in the Liberase + DNase I digestion method.

Comparison with existing methods

The AO/PI staining method was employed to assess the quality of retinal single-cell suspensions. T cells and their subpopulations in retinal tissues were analyzed by flow cytometry. These results were integrated to evaluate the optimal preparation protocol for retinal single-cell suspensions.

Conclusions

Papain digestion is a superior method for preparing retinal single-cell suspensions.

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大鼠视网膜单细胞悬浮液制备方法与流式细胞术免疫分型方法的比较。

背景：虽然眼睛传统上被认为是一个具有免疫特权的器官，但越来越多的研究重新评估了免疫反应在视网膜病变中的作用。应用流式细胞术对视网膜单细胞悬液进行免疫表型分析已逐渐引起人们的重视。新方法：分别采用胰蛋白酶消化法、木瓜蛋白酶消化法、机械研磨法和liberase + DNaseⅠ消化法分离视网膜组织。随后，我们用台盼蓝（TPB）和吖啶橙/碘化丙啶（AO/PI）两种不同的染料评估细胞悬浮液的质量（结块率、浓度和活力）。我们用流式细胞术区分T细胞及其亚群。此外，我们还分析了四种制备视网膜单细胞悬液的方法的免疫反应。结果：AO/PI染色法能够快速、准确地评价视网膜单细胞悬液的细胞质量，而TPB染色法存在局限性。流式细胞分析显示，分散在木瓜蛋白酶和胰蛋白酶中的单细胞悬浮液显示出细胞粘附性降低。然而，胰蛋白酶消化可能影响抗体结合。机械研磨处理降低了细胞产量，容易产生双阳性反应。在Liberase + DNase I酶切法中，细胞环门内的细胞数量受到明显限制。与现有方法比较：采用AO/PI染色法评价视网膜单细胞悬液的质量。用流式细胞术分析视网膜组织中的T细胞及其亚群。综合这些结果来评估视网膜单细胞悬液的最佳制备方案。结论：木瓜蛋白酶消化法是制备视网膜单细胞悬液的优良方法。

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来源期刊

Journal of Neuroscience Methods 医学-神经科学

CiteScore

7.10

自引率

3.30%

发文量

226

审稿时长

52 days

期刊介绍： The Journal of Neuroscience Methods publishes papers that describe new methods that are specifically for neuroscience research conducted in invertebrates, vertebrates or in man. Major methodological improvements or important refinements of established neuroscience methods are also considered for publication. The Journal''s Scope includes all aspects of contemporary neuroscience research, including anatomical, behavioural, biochemical, cellular, computational, molecular, invasive and non-invasive imaging, optogenetic, and physiological research investigations.