preparing single-cell suspension from mouse brain to study immune response to brain disorders

从小鼠脑和颅骨骨髓中获得单细胞悬液

The brain, the central hub of the nervous system, is protected by the skull. Beneath the skull are three layers of connective tissue known as the meninges - the dura mater, arachnoid mater, and pia mater. Cerebrospinal fluid (CSF) circulates in the subarachnoid space between the arachnoid mater and pia mater, cushioning the brain and also removing waste via the glymphatic system1,2. Together, this unique architecture provides a secure and supportive environment that maintains the stability of the brain and shields it from potential injury.
The brain has long been considered immune-privileged. However, this notion has been partially abandoned as mounting evidence indicates that, in addition to resident microglia in the parenchyma, the borders of the brain, including the choroid plexus and meninges, host a diverse array of immune cells3. These cells play critical roles in maintaining homeostasis, surveilling brain health, and initiating the immune response to brain injury. Notably, recent findings indicate that the skull is involved in meninges immunity, and may contribute to the immune response in the brain after injury. In 2018, Herisson et al. made a seminal discovery of direct vascular channels that link the skull bone marrow to the meninges, thereby establishing an anatomical route for leukocyte migration4,5. Later, Cugurra et al. demonstrated that many myeloid cells (e.g., monocytes and neutrophils) and B cells in the meninges do not originate from the blood6. Using techniques such as calvaria bone-flap transplantation and selective irradiation regimens, the authors provided compelling evidence that the skull bone marrow serves as a local source for myeloid cells in the meninges as well as CNS parenchyma after CNS injury6. Further, another study proposed that meningeal B cells are constantly supplied by the skull bone marrow7. More recently, a novel structure, termed the arachnoid cuff exit (ACE), has been identified as a direct gateway between the dura mater and the brain for immune cell trafficking8.
These exciting findings have important implications for the origin of infiltrating immune cells into the injured brain (e.g., after ischemic stroke). A large body of evidence has indicated that after stroke, many immune cells infiltrate the brain, contributing to both acute brain damage and chronic brain recovery. The conventional notion&#160;is that these cells are circulating leukocytes in the blood that infiltrate the brain, which is largely facilitated by stroke-induced blood-brain barrier damage. However, this notion has been challenged. In one study, immune cells in the skull and the tibia of mice were labeled differently, and at 6 h after stroke, a significantly greater decrease in neutrophils and monocytes was found in the skull vs. the tibia, and more skull-derived neutrophils were present in the ischemic brain. These data suggest that in the acute stroke phase, neutrophils in the ischemic brain primarily originate from the skull bone marrow4. Interestingly, CSF may guide this migration. Indeed, two recent reports demonstrated that CSF can directly relay signaling cues from the brain into the skull bone marrow via skull channels, and instruct cell migration and hematopoiesis in the skull bone marrow after CNS injury9,10.
In light of these recent findings, it has become important to analyze changes in immune cells in both the brain and the skull bone marrow, when studying the immune response to brain disorders. In such investigations, sufficient numbers of high-quality immune cells are needed for downstream analyses such as cell sorting, flow cytometry analysis, and single-cell RNA sequencing (scRNA-seq). Here, the overall goal is to present two optimized procedures for preparing single-cell suspensions from brain tissue and skull bone marrow. It is important to note that the calvaria (frontal bone, occipital bone, and parietal bones) of the skull are typically used to extract bone marrow, and this bone marrow is specifically referred to as skull bone marrow throughout this study.

作者聚焦：研究脑缺血后的复杂免疫反应

从小鼠大脑和颅骨中分离免疫细胞

制备小鼠大脑的单细胞悬液以研究对脑部疾病的免疫反应

为了从分离的神经元脑中获得单细胞悬液，将大脑置于预冷的 15 毫升玻璃 Dounce 匀浆器中，该匀浆含有适量冰冷的 Hanks 平衡盐溶液或 HBSS 缓冲液。使用松散的 Dounce 杵在冰上轻轻缓慢地敲击约 100 至 120 次，使脑组织分离。通过 50 毫升离心管中的 70 微米细胞过滤器过滤所得脑组织悬液。用 5 毫升 HBSS 冲洗 Dounce 管并继续过滤以最大限度地收集细胞。将过滤的组织悬液在 4 摄氏度下以 550 g 离心 6 分钟。使用真空抽吸器去除上清液，并将细胞上颚重悬于 1 毫升 30% 等渗密度梯度溶液中。将细胞悬液转移到 15 mL离心管中。加入适量的 30% 密度梯度溶液，轻轻倒置试管，确保充分混合。立即以 845 g 离心管，在 4 摄氏度下加速和制动设置为 3 级 20 分钟。完成后，轻轻地从离心机中取出试管，不要摇晃，然后使用连接到真空的 1 毫升尖端小心吸出髓鞘上层。然后吸出上清液，留下 1 到 2 毫升，并用至少 10 毫升的冷 HBSS 重悬细胞上颚。在 4 摄氏度下以 550 g 离心 6 分钟。用至少 7 mL 的冷 HBSS 再洗涤一次，然后再次离心。去除尽可能多的上清液后，将细胞重悬于 100 至 200 μL 流式细胞术缓冲液中，用于流式细胞术分析。

preparing-single-cell-suspension-from-mouse-brain-to-study-immune

Research

JoVE Journal

Neuroscience

Preparing Single-Cell Suspension from Mouse Brain to Study Immune Response to Brain Disorders

246 次观看. 为了从分离的神经元脑中获得单细胞悬液，将大脑置于预冷的 15 毫升玻璃 Dounce 匀浆器中，该匀浆含有适量冰冷的 Hanks 平衡盐溶液或 HBSS 缓冲液。使用松散的 Dounce 杵在冰上轻轻缓慢地敲击约 100 至 120 次，使脑组织分离。通过 50 毫升离心管中的 70 微米细胞过滤器过滤所得脑组织悬液。用 5 毫升 HBSS 冲洗 Dounce 管并继续过滤以最大限度地收集细胞。将过滤的组织悬液在 4...

视频: 制备小鼠大脑的单细胞悬液以研究对脑部疾病的免疫反应

Isolating Immune Cells from Mouse Brain and Skull

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only in the brain, but also in the skull. A key step toward analyzing changes in immune cell populations in both the brain and skull bone marrow after brain damage (e.g., stroke) is to obtain sufficient numbers of high-quality immune cells for downstream analyses. Here, two optimized protocols are provided for isolating immune cells from the brain and skull bone marrow. The advantages of both protocols are reflected in their simplicity, speed, and efficacy in yielding a large quantity of viable immune cells. These cells may be suitable for a range of downstream applications, such as cell sorting, flow cytometry, and transcriptomic analysis. To demonstrate the effectiveness of the protocols, immunophenotyping experiments were performed on stroke brains and normal brain skull bone marrow using flow cytometry analysis, and the results aligned with findings from published studies.

To investigate the immune response to brain disorders, one common approach is to analyze changes in immune cells. Here, two simple and effective protocols are provided for isolating immune cells from murine brain tissue and skull bone marrow.

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only ...