Thanks to Yanyang Bai for helping with the immunoblot in Figure 5. This work was supported by the Canadian Institutes for Health Research [CRC-RS 950-232402 to AC]; Natural Sciences and Engineering Research Council of Canada [RGPIN-2019-04450, DGECR-2019-00069 to AC]; Scottish Rite Charitable Foundation [21103 to AC] and the Brain Canada Foundation [AWD-023132 to AC]; University of British Columbia Aboriginal Graduate Fellowship (6481 to MT); British Columbia Graduate Scholarship (6768 to MT); Canadian Open Neuroscience Platform Student Scholar Award (10901 to JK); University of British Columbia Four Year Doctoral Fellowship (6569 to JK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Mouse Brain Histone Modification Quantification Using Flow Cytometry

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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+histone+modification+patterns+predict+risk+of+prostate+cancer+recurrence.">Global histone modification patterns predict risk of prostate cancer recurrence.</a> Nature. 435 (7046), 1262-1266 (2005).</li><li>Liu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Histone+Modification+Patterns+as+Prognostic+Markers+to+Classify+Glioma+Patients.">Global Histone Modification Patterns as Prognostic Markers to Classify Glioma Patients.</a> Cancer Epidemiology, Biomarkers &amp; Prevention. 19 (11), 2888-2896 (2010).</li><li>Pan, R. 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The authors have nothing to disclose.

The protocol presented enables quantitative assessment of global HPTM levels through flow cytometry. While this protocol presents a novel method, previous studies have done quantitative assessment of proteins using a similar approach26. Previous methods used to assess global levels of HPTMs include immunohistochemistry and western blot16,17,19,20. The flow cytometry-based method presented is&#160;an&#160;easily quantifiable method, whereas western blot and immunohistochemistry are semi-quantitative and have lower throughput. Western blot relies on cell lysis and thus requires both protein normalization and a loading control protein that is assumed to be unchanged by the experimental condition27. Immunohistochemistry is semiquantitative and very low throughput as it is difficult to quantitatively assess the amount of protein without examining on a single cell level16. Similarly, for the isolated microglia, there is a benefit to using the flow cytometry method due to the limited yield as western blot requires much larger protein input19. The low cell number requirements allow for multiple staining panels to be run from the same animal.
However, as with any other method, there are limitations to this technique including antibody cost and availability, as not all antibodies work well in a flow cytometry setting. In addition, compared to immunoblot, the concentration of antibody required is much higher. While multiplexing allows for multiple antibodies to be used on the same panel of cells, cells cannot be stripped of the antibody after analysis, thus limiting the cell usage to one per antibody species. This is different from immunoblot in which the same blot can be used repeatedly. However, depending on the availability of antibodies and the number of detection channels on a cytometer, it would be possible to examine up to a dozen marks simultaneously.
The current method captures only global levels of protein expression and not the specific genomic location, and changes in global levels may not reflect changes at individual genomic loci. Similarly, a lack of change in global levels may not mean that no genomic loci are undergoing changes, simply that the global changes sum to no differences between treatments. As such, this technique is meant to be used as a screen to identify HPTMs of interest for genomic analysis. In addition, this method does not allow for comparison across different protein marks except for when assessed as a fold change to control. Therefore, this is limited compared to a standard curve-based method such as ELISA for protein determination.
The protocol presented offers a strategy for isolating live brain microglia. This protocol relies on P2RY12 protein expression for microglia isolation. However, P2RY12 is a homeostatic marker in microglia and can be downregulated in disease models, such as 5XFAD22. Therefore, when using a disease model animal be sure to choose other marker proteins such as TMEM119, CD11b, or CD45 to aid in isolation of microglia23. Similarly, we present this protocol as isolation from the hippocampus and/or the cortex. This protocol would work to isolate microglia from other brain regions including white matter regions, however, multiple animals may be required to obtain enough microglia depending on the size of the regions of interest.
The protocol presented can robustly isolate live, brain microglia, but there are several steps, described below, in the isolation stage that can decrease cell yield if performed incorrectly.
Perfusions for this protocol result in a higher percentage of microglia in the immune enriched fragment which will reduce the amount of time at the sorter. However, perfusion is not required, and other methods of euthanasia can be used if required.
During microglia isolation, myelin should be completely removed. Flow cytometers rely on cells being able to travel through narrow tubing at a rapid pace. Due to its viscosity and tendency to clump, myelin causes problems with cytometers, often causing clogs which can both damage the equipment and destroy the sample, reducing yield drastically. Be cautious to remove all of the myelin during collection of immune-enriched fragments to avoid having issues downstream.
Plate staining versus tube staining: In this protocol, we described two options for staining cells in either 1.5 mL tubes or a 96 well plate. The use case for each depends on the experiment; however generally tube staining is lower risk for impacting yield than plate staining as the flick risks loss of cells if done incorrectly. Plate staining is much faster as aspirating the supernatant for each tube is time consuming. Prior to fixation (for sorting, etc.), use tube staining to maximize yield and reduce risk of loss. However, for HPTM analysis, once cells are fixed for intranuclear staining, the pellet is more stable, and there is reduced risk of loss with flicking.
Establishing the discontinuous density gradient: When establishing the layering, setting up the layers properly is essential to obtain the immune enriched fraction. If the layers are disturbed or mixed and appear cloudy, the cells will not sort to their desired location, and there will be difficulty in obtaining the immune-enriched cell fraction. If this occurs, spin with the density medium to remove myelin and then collect the entire remaining fractions, dilute with 3 mL of FACS buffer to 1 mL of density medium and mix well (this will require multiple tubes). Spin at 500 x g for 10 min with the brake on 0. Discard the supernatant, leaving only ~300 &#181;L solution. Collect the entire sample and stain. This will yield in reduced sort percentages and a higher amount of time spent at the cytometer, but the yield can still be comparable.
When using the isolation method, it is beneficial to be able to collect cells for RNA and for HPTM evaluation from the same mouse brain. In this situation, after sorting the live microglia, cells can be divided to allocate a portion to RNA evaluation (minimum input number of cells to obtain a decent RNA yield is 75,000 cells) and a portion for further flow cytometry analysis (minimum 10,000 cells per well for good determination of MFI). In this case, flow cytometer sorting is required. However, when only planning to use the cells for HPTM analysis, sorting is not required, and the immune fraction can be stained with the P2RY12 antibody and HPTM antibody. Gating on the cytometer can then be set for P2RY12+ microglia, as would be done for flow sorting, to analyze only HPTM signal within microglia. Eliminating the sorting allows for the protocol to be faster and more cost effective. In addition, if evaluating HPTMs from cultured cells, starting at the staining protocol is sufficient and no cell marker antibodies are required as demonstrated in Figure 6. The HPTM evaluation protocol can be used for many cell types including cultured, primary, and IPSC derived cells.
Finally, while we have presented only two potential uses of microglia downstream of isolation, there are many others including epigenetic techniques such as ChIP, CUT&#38;Tag and CUT&#38;RUN. In the case of genomic epigenetic techniques, where characterizing changes at specific loci is of interest, choose specific inhibitors for writers and erasers of chromatin marks11 tailored to the experiments to ensure that any microglial epigenetic modifications profiled are not technical artifacts from any steps in the isolation procedure such as enzymatic digestion. When assessing changes in global levels of epigenetic marks, such as by using quantitative flow cytometry, any procedure-induced changes are not expected to be so large that they are detected at the global level.
Overall, the discussed methods provide a novel, single cell method for quantifying global levels of histone modifications and other epigenetic changes by flow cytometry. We demonstrated that this method is sufficiently sensitive to detect global changes in the enhancer marker H3K27ac in microglia in response to LPS in vivo. This is consistent with previous ChIP-sequencing of H3K27ac following LPS stimulation showing dramatic remodeling of enhancers responsive to LPS28. Applications of this method will allow for examination of global epigenetic changes across different brain cell types in development and disease.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5M EDTA</td>
			<td>Invitrogen</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Falcon Centrifuge Tubes, Polypropylene, Sterile&#160;</td>
			<td>Falcon</td>
			<td>352196</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well Clear Not Treated Plates</td>
			<td>Costar</td>
			<td>3738</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well Clear Polystyrene Microplate, clear round bottom, non treated surface</td>
			<td>Corning</td>
			<td>3788</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetyl Histone 3 K9 (C5B11)</td>
			<td>Cell Signalling Technology</td>
			<td>9649S</td>
			<td>Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Acetyl Histone H4 K8 (2594)</td>
			<td>Cell Signalling Technology</td>
			<td>2594S</td>
			<td>Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Acetyl-Histone H3 K27 (D5E4)</td>
			<td>Cell Signalling Technology</td>
			<td>8173S</td>
			<td>Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Acetyl-Histone H3 Lys27 (MA523516)</td>
			<td>Invitrogen</td>
			<td>MA523516</td>
			<td>Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Actinomycin D</td>
			<td>New England Biolabs</td>
			<td>15021S</td>
			<td></td>
		</tr>
		<tr>
			<td>Anisomycin</td>
			<td>New England Biolabs</td>
			<td>2222S</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Histone H3 (tri methyl K4)&#160;</td>
			<td>Abcam</td>
			<td>ab213224</td>
			<td>Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Anti-Lactyl-Histone H4 (Lys 12) Rabbit mAb</td>
			<td>PTM Biolabs</td>
			<td>PTM-1411RM</td>
			<td>Dilution: 1:250</td>
		</tr>
		<tr>
			<td>Anti-L-Lactyllysine Rabbit pAb</td>
			<td>PRM Biolabs</td>
			<td>PTM-1401RM</td>
			<td>Dilution: 1:250</td>
		</tr>
		<tr>
			<td>Apc anti-P2RY12 Antibody, Clone: S16007D</td>
			<td>BioLegend</td>
			<td>848006</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Tocris</td>
			<td>5217</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyto-Last Buffer</td>
			<td>BioLegend</td>
			<td>422501</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethylsulfoxide, sterile</td>
			<td>Cell Signalling Technology</td>
			<td>12611S</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>STEMCELL Technologies</td>
			<td>07900</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti Mouse AlexaFluor488</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-546-150</td>
			<td>Dilution: 1:500</td>
		</tr>
		<tr>
			<td>Donkey Anti Rabbit AlexaFluor488</td>
			<td>ABclonal</td>
			<td>AS035</td>
			<td>Dilution: 1:500</td>
		</tr>
		<tr>
			<td>Donkey Anti Rabbit AlexaFluor568</td>
			<td>Invitrogen</td>
			<td>A10042</td>
			<td>Dilution: 1:500</td>
		</tr>
		<tr>
			<td>Donkey Anti Rabbit Brilliant Violet 421</td>
			<td>BioLegend</td>
			<td>406410</td>
			<td>Dilution: 1:500</td>
		</tr>
		<tr>
			<td>Fisherbrand Disposable Graduated Transfer Pipettes</td>
			<td>Fisherbrand</td>
			<td>13-711-9AM</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Disposable PES Filter Unit, 250mL&#160;</td>
			<td>Fisherbrand</td>
			<td>FB12566502</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K18ac Polyclonal Antibody</td>
			<td>Invitrogen</td>
			<td>720095</td>
			<td>Dilution: 1:100</td>
		</tr>
		<tr>
			<td>HBSS (10X), no calcium, no magnesium, no phenol red</td>
			<td>Gibco</td>
			<td>14185052</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red</td>
			<td>Gibco</td>
			<td>14175103</td>
			<td></td>
		</tr>
		<tr>
			<td>Histone 3 Trimethyl K27 (ab6002)</td>
			<td>Abcam</td>
			<td>ab6002</td>
			<td>Dilution: 1:100</td>
		</tr>
		<tr>
			<td>KONTES Dounce Tissue Grinders 125mm 7mL&#160;</td>
			<td>VWR</td>
			<td>885300-0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactyl-Histone H3 (Lys 18) Rabbit mAb</td>
			<td>PTM BIolabs</td>
			<td>PTM-1406RM</td>
			<td>Dilution: 1:250</td>
		</tr>
		<tr>
			<td>Lipopolysacharide&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>L5418</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>OneComp eBeads Compensation Beads</td>
			<td>Invitrogen</td>
			<td>01-1111-41</td>
			<td></td>
		</tr>
		<tr>
			<td>PDS Kit, Papain Vial - Worthington Biochemical</td>
			<td>Cedarlane</td>
			<td>LK003178</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>Sigma-Aldrich</td>
			<td>GE17-0891-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>VWR</td>
			<td>RC57004</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAshredder</td>
			<td>Qiagen&#160;</td>
			<td>79656</td>
			<td></td>
		</tr>
		<tr>
			<td>Rainbow Fluorescent Particles, 1 peak (3.0-3.4 uM - Mid Range Intensity</td>
			<td>BioLegend</td>
			<td>422905</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free Microfuge Tubes, 1.5 mL</td>
			<td>Invitrogen</td>
			<td>AM12400</td>
			<td></td>
		</tr>
		<tr>
			<td>Rneasy Plus Micro Kit</td>
			<td>Qiagen&#160;</td>
			<td>74034</td>
			<td></td>
		</tr>
		<tr>
			<td>Round Bottom Polypropylene Tubes with Caps, 5 mL</td>
			<td>Corning</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>Triptolide</td>
			<td>New England Biolabs</td>
			<td>97539</td>
			<td></td>
		</tr>
		<tr>
			<td>True Nuclear Transcription Factor Buffer Set</td>
			<td>BioLegend</td>
			<td>424401</td>
			<td></td>
		</tr>
		<tr>
			<td>TruStain FcX PLUS (anti-mouse CD16/32) Antibody</td>
			<td>BioLegend</td>
			<td>156604</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>VWR</td>
			<td>97063-702</td>
			<td></td>
		</tr>
		<tr>
			<td>Zombie Aqua Fixable Viability Kit</td>
			<td>BioLegend</td>
			<td>423102</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Author Spotlight: Enhancements in Gene Expression Regulation Research

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

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.

Adult mice were transcardially perfused and sacrificed for microglia isolation. Microglia were isolated on ice and stained with P2RY12-APC and violet 525 live dead antibodies. Cells that were determined to be positive for P2RY12 and negative for violet 525 live dead stain were sorted as live microglia. The average yield of microglia from a dissected mouse brain was 1.28 x 105 &#177; 0.05 (mean &#177; standard error of mean (SEM), N=100). There is no difference in the yield of microglia from female (1.25 x 105 &#177; 0.09 [mean &#177; SEM, N=46]) and male (1.32 x 105 &#177; 0.07 [mean &#177; SEM, N=54]) mice (t(98)=0.6365, p=0.526). When isolating from specific brain regions, the average yield of microglia from mouse cortices is 8.3 x 104 &#177;&#160; 0.08 (mean &#177; SEM, N=15) and from mouse hippocampus is 4.1 x 104 &#177; 0.02 (mean &#177; SEM, N=16). As expected, there is a significant difference in the yield of microglia from each brain region (F(2, 128)=25.25, P&#60;0.0001). Following microglia isolation, RNA was extracted from the isolated cells using a low-input RNA isolation kit. Consistently the RNA integrity score (RIN) was above 9.0 (9.62&#160;&#177; 0.05) and the average yield of RNA per cell was 0.25 &#177; 0.01 pg (mean &#177; SEM, N=32; Supplementary File S2).
Adult mice were intraperitoneally injected with 1 mg/kg lipopolysaccharide (LPS) 24 h prior to sacrifice. The mice were transcardially perfused with HBSS, and microglia isolated from the whole brain according to the described protocol (Figure 5A). For each stain, 20,000-30,000 cells were allocated to each panel of antibodies. The global levels of histone 3 lysine 27 acetylation (H3K27Ac) were assessed in isolated microglia via flow cytometry. For male and female mice, LPS treatment induced increase in H3K27Ac when the MFI is normalized within sex (t(6)=9.676, p&#60;0.0001; Figure 5B). When examining the histograms for the stained cells, the populations remain normally distributed with similar variation; however, the cells have shifted to increased fluorescence resulting in the increase in MFI (Figure 5C). When examining H3K9Ac in the same treatment, there is a similar increase in H3K9Ac (t(6)=7.299, p=0.0003; Figure 5D,E) however the fold change of LPS relative to PBS of H3K9Ac signal is less than H3K27Ac signal.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65080/65080fig05.jpg" /> 
Figure 5: Global changes in histone acetylation in isolated microglia. (A) Mice are injected intraperitoneally with phosphate buffered saline (PBS) or 1 mg/kg lipopolysaccharide (LPS) 24 h prior to sacrifice. The microglia are collected from the immune enriched fraction and fixed for flow cytometry and global histone post translational modification assessment. The median fluorescent intensity is assessed as a proxy for protein expression. Created with BioRender.com. (B) Global levels of H3K27Ac increased in response to LPS treatment. Fold change to PBS normalized within experiment and sex. Unpaired two tailed t-test, t(6)=9.676, p&#60;0.0001. Bar graph depicts the mean &#177; SEM. N=8 animals; 2 per condition in 2 independent experiments. (C) Example histograms depicting shift of H3K27Ac fluorescent intensity. Modal depicts histograms from PBS-injected versus LPS-injected mice. (D) Example histograms depicting shift of H3K9Ac fluorescent intensity. Modal depicts histograms from PBS-injected versus LPS-injected mice. (E) Global levels of H3K9Ac increased in response to LPS treatment. Fold change to PBS normalized within experiment and sex. Unpaired two tailed t-test, t(6)=7.299, p=0.0003. Bar graph depicts the mean &#177; SEM. N=8 animals; 2 per condition in 2 independent experiments. <a href="https://www.jove.com/files/ftp_upload/65080/65080fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To confirm that the method described was comparable to other previously used methods for global histone modification quantification, we aimed to use immunoblot as a comparative tool. However, the yield from the isolated microglia is simply too low to enable reasonable assessment. Therefore, we used cultured BV2 cells to compare the intracellular flow cytometry method to a Western blot (WB). BV2 cells were grown in complete media (DMEMF12, 10% FBS, 1x penicillin/streptamycin, and 1x L-glutamine) at 37 &#176;C, 5% CO2. Cells were passaged with 0.25% trypsin-EDTA and plated at a density of 250,000 cells/well and treated in reduced serum media (DMEM F12, 2% FBS, 1x penicillin/streptamycin, and 1x L-glutamine) and allowed to recover for 12 h at 37 &#176;C, 5% CO2. Cells were treated with 25 ng/mL LPS for 24 h prior to fixation as described above or lysis with a WB lysis buffer. Signal of H3K27Ac was performed by both methods with GAPDH used as a loading control for WB. Analysis of the normalized fluorescent intensity compared to the PBS control was determined for each group (Figure 6A). When examining the change in normalized H3K27Ac signal by WB, there was a 1.527-fold increase in the LPS treated condition relative to the H2O control which was determined to be significant by unpaired t-test (t=3.024, df=5; p=0.0293). When examining the change using flow cytometry, there was a 1.482-fold increase in the LPS treated condition which was determined to be significant (t=7.843, df=10; p&#60;0.0001). Using a 2-way ANOVA to compare the methods, there was determined to be a significant effect of the treatment (F(1,15)=45.21,p&#60;0.0001), but not the method (F(1,15)=0.05545, p=0.8697) or interaction (F(1,15)=0.02785, p=0.8697). In addition, we verify here that there is no change in histone H3 levels by both Western blot and flow cytometry as 2-way ANOVA revealed no significant effect of the LPS treatment (F(1,7)=0.02170,p=0.8870), the method (F(1,7)=0.01191, p=0.9162) or the interaction (F(1,7=0.01191, p=0.9162; Figure 6B). Example blots and histogram shifts for this data are shown as well (Figure 6C,D).
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65080/65080fig06.jpg" /> 
Figure 6: Method comparison for quantification of global histone modification change between flow cytometry and western blot. (A) BV2 cells are treated with 25 ng/mL lipopolysaccharide (LPS) or H2O for 24 h prior to analysis. The fluorescent intensity of H3K27Ac is depicted as fold change to the vehicle control, phosphate buffered saline (PBS), for both flow cytometry and western blot. 2-way ANOVA revealed significant effect of the LPS treatment (F(1,15)=45.21, p&#60;0.0001), but not the method (F(1,15)=0.05545, p=0.8697) or interaction (F(1,15)=0.02785, p=0.8697). Tukey&#39;s correction for multiple hypothesis testing was applied for the residuals. * presents 0.0332, ** presents 0.0021. (B) The fluorescent intensity for histone H3&#160;is depicted as fold change to PBS for both flow cytometry and western blot. 2-way ANOVA revealed no significant effect of the LPS treatment (F(1,7)=0.02170, p=0.8870) or the method (F(1,7)=0.01191, p=0.9162) or the interaction (F(1,7=0.01191, p=0.9162). (C) Example blots and (D) flow cytometry shifts are portrayed. Histogram size is normalized to percent based on number of cells present at the mode fluorescent intensity. Bar graph depicts the mean SEM. n=2 independent experiments, 2 per condition per experiment. <a href="https://www.jove.com/files/ftp_upload/65080/65080fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
All together, these results show that this technique can be used to quantitatively assess the global HPTM levels in isolated microglia. In addition, the method was shown to be comparable to previous techniques but requiring much lower cell inputs. In addition, while not shown, with proper compensation, the present technique can be used with multiple antibodies on the same panel assessing different HPTMs.
Supplementary file S1: Example analysis files. This file contains a wsp analysis file and 7 fcs files including the no stain, P2RY12FMO, 568FMO, two PBS treated animals and two LPS treated animals stained with H3K27Ac. The purpose of this file is to demonstrate the analysis and gating on an experiment that could depict what a successful experiment looked like. <a href="https://www.jove.com/files/ftp_upload/65080/SupplementalS1.zip" target="_blank">Please click here to download this File.</a>
Supplementary File S2: Isolation data. The file included contains the relevant data post microglia sort which contains the microglia and RNA yield from the described protocol. <a href="https://www.jove.com/files/ftp_upload/65080/SupplementalFileS2.xlsx" target="_blank">Please click here to download this File.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>GATED POPULATION&#160;</td>
			<td>Frequency of Parent</td>
			<td>Frequency of Total</td>
			<td>Count</td>
		</tr>
		<tr>
			<td>S1</td>
			<td>89.00%</td>
			<td>89.00%</td>
			<td>25672</td>
		</tr>
		<tr>
			<td>S2&#62;S1</td>
			<td>88.73%</td>
			<td>78.97%</td>
			<td>22779</td>
		</tr>
		<tr>
			<td>APC+ &#62; S2 &#62; S1&#160;</td>
			<td>76.61%</td>
			<td>60.50%</td>
			<td>17452</td>
		</tr>
		<tr>
			<td>610+ &#62; APC+ &#62; S2 &#62; S1</td>
			<td>99.56%</td>
			<td>60.24%</td>
			<td>17376</td>
		</tr>
	</tbody>
</table>
Table 2: Example sample lineage chart depicts percentage and event numbers required for accurate protein detection.

Watch this Scientific Journal Video about Author Spotlight: Enhancements in Gene Expression Regulation Research at JoVE.com

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 ...

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Research

JoVE Journal

Neuroscience

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.

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.

Mouse Brain Enriched Immune Pellet Plate Staining Method for Histone Quantification

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.

616 Views.  University of British Columbia.  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.

Mouse Brain Histone Modification Quantification Using Flow Cytometry

Summary