mouse brain enriched immune pellet plate staining method for histone quantification

フローサイトメトリーを用いたマウス脳ヒストン修飾定量

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.

著者スポットライト:遺伝子発現制御研究の強化

単離マウス脳ミクログリアにおける核内フローサイトメトリーを用いたグローバルヒストン翻訳後修飾の定量化

Our research aims to understand the fundamental regulatory mechanisms controlling gene expression in the brain, and we're particularly interested in understanding how disruption of these mechanisms contributes to neurodevelopmental and neuropsychiatric conditions. We focus on understanding how genetic and environmental risk factors combined to alter cellular function and produce behavioral impairments. This protocol includes methods for high-throughput screening of histone modifications in isolated microglia, allowing us to screen through dozens of different epigenetic modifications before committing to high-throughput sequencing, and functional follow-up studies.Our protocol provides measures of global histo modifications similar to Western plot. However, our technique is both faster, requires fewer input cells, is more quantitative, and can be multiplexed.

Watch this Scientific Journal Video about Mouse Brain Enriched Immune Pellet Plate Staining Method for Histone Quantification at JoVE.com

マウス脳強化免疫ペレットプレート染色法によるヒストン定量化

マウスの脳に富ませた免疫ペレットを15ミリリットルのチューブから丸底の96ウェルプレートに移します。プレートを500Gで5分間遠心分離した後、フリックして上澄みを取り除きます。細胞を100マイクロリットルのFACS緩衝液に再懸濁し、各ウェルから10マイクロリットルをコントロールウェルに移します。500 Gで5分間遠心分離します。遠心分離した細胞から上清を除去した後、50マイクロリットルの二重濃度FCブロックを添加する。氷上で10分間インキュベートします。次に、50マイクロリットルの抗体マスターミックスを最終的な所望濃度の2倍で添加し、暗所の氷上でインキュベートします。次に、200マイクロリットルのFACSバッファーを各ウェルに加え、500 Gで5分間遠心分離します。上清をフリックして廃棄し、300マイクロリットルのFACS緩衝液で細胞を洗浄します。細胞を遠心分離し、200マイクロリットルのFACS緩衝液に再懸濁します。最後に、懸濁液を300マイクロリットルのFACS緩衝液が入ったフローソートチューブに移します。

mouse-brain-enriched-immune-pellet-plate-staining-method-for-histone

Research

JoVE Journal

Neuroscience

Mouse Brain Enriched Immune Pellet Plate Staining Method for Histone Quantification

174 ビュー.  University of British Columbia. マウスの脳に富ませた免疫ペレットを15ミリリットルのチューブから丸底の96ウェルプレートに移します。プレートを500Gで5分間遠心分離した後、フリックして上澄みを取り除きます。細胞を100マイクロリットルのFACS緩衝液に再懸濁し、各ウェルから10マイクロリットルをコントロールウェルに移します。500 Gで5分間遠心分離します。遠心分離した細胞から上清を除?...

ビデオ: マウス脳強化免疫ペレットプレート染色法によるヒストン定量化

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 ...

神経科学

Mouse Brain Dissection for Microglia Isolation

Begin by placing the isolated mouse brain tissue in 1 milliliter of digestion buffer per brain in separate Petri dishes on ice. Thoroughly chop the brain tissues into small pieces using a clean scalpel blade. Using a plastic transfer pipette with its tip cutoff, carefully transfer minced brains into individual wells inside a 24-well plate on ice.Cover the plate with a large piece of transparent, flexible film, close the lid, and incubate on ice for 30 minutes. Next, transfer the digested brain solution into individual 7-milliliter iced glass dounce homogenizers on ice containing 5 milliliters of cold FACS buffer. Gently dounce each brain with a loose pestle until a single-cell suspension is obtained.Softly dounce with the tight pestle three to four times to ensure a single-cell suspension before transferring to 15-milliliter polypropylene tubes.

ミクログリア単離のためのマウス脳解剖

Mouse Brain Immune-Enriched Fragment Isolation for Histone PTM Quantification

Begin by adding 2.125 milliliters of isotonic density gradient to a 15 milliliter polypropylene tube containing murine brain homogenate. Top up each tube with fax buffer to a final volume of 8.5 milliliters. Gently invert the tubes 20 times to mix thoroughly.Using a narrow graduated transfer pipette, gently underlay four milliliters of pink, 37%density gradient establishing two clean layers. Switch transfer pipettes and underlay two milliliters of the blue, 70%density gradient below the 37%layer. Transfer the tubes to a centrifuge cool to four degrees Celsius and spin them at 500 G for 20 minutes with the braking ramp set to the lowest setting.After centrifugation, discard the myelin from the top of the 15 milliliter tube using a clean transfer pipette. Carefully collect the top fragment of the density gradient into a clean 15 milliliter polypropylene tube using another transfer pipette. Next, gather all cells in the sample by slowly circling the pipette along the sides of the tube while collecting the immune enriched fragment.Transfer the immune enriched fragment into a new 15 milliliter polypropylene tube. Wash the sample by adding 10 milliliters of fax to the tube. Gently invert the tube 20 times to mix thoroughly.Spin the tubes in a cooled centrifuge with the braking ramp set to the lowest setting to pellet the isolated immune cells.

マウス脳免疫濃縮フラグメント単離によるヒストンPTM定量化

Transfer a mouse brain enriched immune pellet from a 15-milliliter tube to a round bottom 96-well plate. After centrifuging the plate at 500 G for five minutes, Remove the supernatant by flicking. Resuspend the cells in 100 microliters of FACS buffer, then transfer 10 microliters from each well into the control wells.Centrifuge at 500 G for five minutes. After removing the supernatant from the centrifuged cells, add 50 microliters of double concentration FC block. Incubate for 10 minutes on ice.Now add 50 microliters of the antibody master mix at double the final desired concentration and incubate it on ice in the dark. Then, add 200 microliters of the FACS buffer to each well and centrifuge at 500 G for five minutes. Discard the supernatant by flicking, then wash the cells in 300 microliters of FACS buffer.Centrifuge and resuspend the cells in 200 microliters of FACS buffer. Finally, transfer the suspension to a flow sort tube containing 300 microliters of FACS buffer.

Flow Cytometry and Data Analysis of Murine Brain Histone Post-Translational Modifications

Analyze the antibody panel, confirm that the cytometer has a minimum of four lasers, including red, yellow, blue, and violet. Establish the compensation matrix with either compensation beads or single stain cell controls. To calibrate and standardize, run rainbow fluorescent beads at the beginning of each experiment by adjusting the photomultiplier tube voltage until the bead peaks align with the target values from the previous experiments.Set the photomultiplier tube voltage and gain for the experiment. Then use antibody captured compensation beads to establish a compensation matrix. Next, plot the side scatter area on log versus the forward scatter height on linear in a dot plot, gate out debris and select for cell size using the S1 gate, choose singlet cells in a dot plot a forward scatter width versus forward scatter height with S2 gate.For establishing floor four gates, use the relevant FMO for each floor four channel. With single parameter histograms determine the positive signal in each channel and establish the gates accordingly. Carefully record the samples using the established gating strategy.Identify microglia using P2 RY 12 plus signal and determine protein expression in the respective channel for microglia only. Establish analysis gates on the cytometer analysis software user interface by replicating the same gating strategy used during recording. Use the add statistics function to select the median for the population of interest on the compensated channel height.Export the MFI values for the respective channels to a spreadsheet using the table editor for further statistical analysis. Lipopolysaccharide treatment induced an increase in histone three lysine 27 acetylation. When the MFI is normalized within sex, histogram analysis of the stained cells showed normally distributed populations with cell shifts to increased fluorescence.A similar increase in histone three lysine 27 acetylation was seen in the stained cells.