mouse brain dissection for microglia isolation

使用流式细胞术进行小鼠脑组蛋白修饰定量

Epigenetics is the study of the mechanisms that regulate gene expression without altering the underlying DNA sequence. Epigenetic regulation of gene expression is dynamic within cells and can allow for rapid and coordinated responses to various environmental stimuli. The dynamic regulation occurs in part due to changes in the chromatin structure at the level of the nucleosome, which is comprised of histone proteins (H2A, H2B, H3, H4) assembled into an octamer core tightly wound by DNA1. The interactions between the histone proteins and the DNA can control the accessibility of DNA to transcription machinery, which can ultimately control gene expression and other aspects of chromatin biology2. Histone proteins have unstructured tails which feature positively charged residues that form electrostatic interactions with the negatively charged DNA backbone. These interactions result in tight packing of the DNA and reduced DNA accessibility. Covalent modifications to the histone tails, termed histone post-translational modifications (HPTMs), can regulate these interactions3,4. Some of the most well-characterized HPTMs include histone tail acetylation and methylation, which can change the affinity of electrostatic interactions between the histone tails and DNA, resulting in differential accessibility to the underlying DNA and recruitment of transcription factors which recognize these HPTMs at specific sites. HPTMs are regulated by three important classes of enzymes termed readers- which recognize, writers- which deposit, and erasers- which remove HPTMs. Thus, the recruitment or dissolution of reader, writer, or eraser enzymes can ultimately change the landscape of HPTMs and govern the structure and function of chromatin, making their regulation and readout essential for understanding cellular biology and function3,4.
The cells in the central nervous system (CNS) are epigenetically flexible as they change their transcriptome to adapt to environmental stimuli. Accumulating evidence suggests that changes in the epigenome, such as DNA methylation, non-coding RNAs, and HPTMs, play an essential role in memory formation and synaptic function5. Disrupting HPTM dynamics through manipulation of the relevant readers, writers, or erasers can block or enhance associative learning and long-term potentiation6,7,8. Microglia, the resident immune cell of the CNS, rapidly regulate their transcriptome in response to immune stimulation through dynamic changes in their epigenome9,10,11. This high level of adaptation to their local brain environment makes them difficult to examine in an isolated context as studies have shown that the epigenome and transcriptome of microglia becomes altered after only a few hours in culture media after removal from the brain environment11. In addition, as microglia only make up 10% of the brain&#39;s cells, measures examining changes at the whole tissue level lack sensitivity and specificity12,13. As a result, microglia need to be rapidly isolated to examine the epigenetic changes such as HPTM levels, ex vivo.
The methods commonly used to examine HPTMs include chromatin-immunoprecipitation sequencing (ChIP-seq) and cleavage under targets and tagmentation sequencing (CUT&#38;Tag-seq)4. While these techniques are highly specific to an individual HPTM and can inform the presence of HPTMs within a specific genomic context, they can only examine one of the many possible HPTMs within a single experiment11,14 Therefore, before proceeding with such experiments, which require a significant time and money investment, it is highly valuable to narrow down the list of potentially interesting HPTMs for further investigation by first examining changes in global levels of HPTMs. The two main approaches for examining global HPTM levels are immunohistochemistry and western blot analysis, but both approaches are only semi-quantitative, low-throughput, and require large numbers of tissue sections or isolated cells15,16. Thus, we aimed to develop a highly sensitive, quantitative method that could be used to examine the global HPTM levels rapidly and at the single cell level.
The protocol presented enables detection of global HPTM levels rapidly using intranuclear flow cytometry. Previous studies in cancer cells have justified the importance of examining global levels from a clinical perspective17,18. It is also common for studies to use global levels as a screening method prior to assessing genomic location of specific HPTMs of interest19,20. For microglia, assessing global levels following isolation is challenging due to the low cell yield; Pan et al present global HPTM levels from isolated microglia, in which microglia from three animals was pooled to enable protein level detection by western blot19. Using our protocol, we are able to detect global changes with much lower cell inputs, enabling screening of multiple marks per animal and eliminating the need to pool samples.
Here, we describe a protocol to detect HPTM levels rapidly via quantitative intranuclear flow cytometry in isolated microglia. While we focus specifically on HPTM quantification for the sake of brevity, this protocol can be used in the same way to quantify global levels of reader, writer, and eraser enzymes. The protocol is delivered in two parts: first, the isolation method for microglia and, second, the flow cytometry-based method for determining HPTM levels. The isolation method yields cells that can be used for both RNA isolation and HPTM level assessment which allows for the evaluation of gene expression and HPTM levels from the same sample. In addition, the method for HPTM assessment can be used on other cell types as indicated in the protocol.

作者聚焦：基因表达调控研究的增强

使用核内流式细胞术定量分离小鼠脑小胶质细胞中的整体组蛋白翻译后修饰

Watch this Scientific Journal Video about Mouse Brain Dissection for Microglia Isolation at JoVE.com

用于小胶质细胞分离的小鼠脑解剖

首先将分离的小鼠脑组织放入每个大脑1毫升的消化缓冲液中，放在冰上的单独培养皿中。使用干净的手术刀刀片将脑组织彻底切成小块。使用吸头切断的塑料移液管，小心地将切碎的大脑转移到冰上 24 孔板内的单个孔中。用一大块透明柔性薄膜盖住板，盖上盖子，在冰上孵育 30 分钟。接下来，将消化的大脑溶液转移到含有 5 毫升冷 FACS 缓冲液的冰上单独的 7 毫升冰玻璃 dounce 均质器中。用松散的杵轻轻弹动每个大脑，直到获得单细胞悬浮液。在转移到 15 毫升聚丙烯管之前，用紧密的研杵轻轻蘸三到四次以确保单细胞悬浮液。

mouse-brain-dissection-for-microglia-isolation

Research

JoVE Journal

Neuroscience

Mouse Brain Dissection for Microglia Isolation

551 次观看. 首先将分离的小鼠脑组织放入每个大脑1毫升的消化缓冲液中，放在冰上的单独培养皿中。使用干净的手术刀刀片将脑组织彻底切成小块。使用吸头切断的塑料移液管，小心地将切碎的大脑转移到冰上 24 孔板内的单个孔中。用一大块透明柔性薄膜盖住板，盖上盖子，在冰上孵育 30 分钟。接下来，将消化的大脑溶液转移到含有 5 毫升冷 FACS 缓冲液的冰上单独的 7 毫升冰玻?...

视频: 用于小胶质细胞分离的小鼠脑解剖

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...