We would like to thank P. Colosi for EM image preparation. This work was supported by the National Institutes of Health (R01 NS064963, SSC; R01 NS110354, SSC; R01 NS083846, SSC; R21 NS094971, SSC; T32 NS007224, SMT; T32 NS041228, SMT), the United States Department of Defense (W81XWH-17-1-0564, SSC; W81XWH-19-1-0264, VDJ), Aligning Science Across Parkinson&#39;s (ASAP) Collaborative Research Network (SSC), and the Michael J. Fox Foundation Target Advancement Program (MJFF-020160, SSC &#38; VDJ). We created graphical illustrations using BioRender.com.

All experiments with mice were approved by the Institutional Animal Care and Use Committee (IACUC) at Yale University (Protocol 2021-11117) and performed in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Animal care and housing complied with the Guide for the Care and Use of Laboratory Animals33 and were provided by the Yale Animal Resource Center (YARC). Animals were maintained in a 12 h light/dark cycle with ad libitum access to food and water. Five to eight mice or two to four rats per genotype or condition are required for the following protocol. Fewer rats are necessary due to their larger brain volumes. Similarly, the age of the experimental animals may affect fraction yield; additional mice may be required for ages less than 2 months. Otherwise, the outlined procedures apply to both murine species and healthy adult animals of any age. The representative data presented in this study utilized wild-type (C57BL/6J) mice (age = 2 months; four males and four females per replicate) obtained from a commercial source (see Table of Materials).
1. Experimental preparation
NOTE: This protocol requires ~11 h for a single researcher to complete. It is highly recommended to complete benchtop setup (Figure 1), buffer preparation (Table 1), the precooling of centrifuges and rotors to 4 °C, and the collection and labeling of necessary materials and equipment (see Table of Materials) the day prior to protocol execution, where applicable.
<img alt="Molecular biology experiment setup with ice tray, microcentrifuge tubes, pipettes, scissors, and buffer." class="xfigimg" src="/files/ftp_upload/64574/64574fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Benchtop setup. Prior to brain dissections, (A) Dounce glass homogenizers and (B) all buffers were chilled on ice. (C) Protease inhibitor stock solutions were thawed on ice. A second container of wet ice for centrifuge tubes, a Dewar of liquid nitrogen (not shown), and (D) a container of dry ice for short-term storage of the samples flash-frozen in liquid nitrogen were obtained. (E) Microcentrifuge tubes were pre-labeled for all samples, as four aliquots of each subcellular fraction sample per genotype or condition were collected during this procedure (time-saving tip: thoroughly label all the tubes the day before the experiment is performed). (F) An appropriate biohazard waste container, (G) 70% ethanol, (H) surgical tools, and (I) an absorbent surface pad. The required centrifuge tubes and disposables were set aside for efficient access during protocol implementation (not shown). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64574/64574fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Prepare the benchtop for surgery and collect the scissors and forceps required for brain excision (see Table of Materials). Pre-label 1.5 mL microcentrifuge tubes for mouse tail biopsies and four tubes per collected fraction, as outlined in Figure 2.</li>
	<li>Obtain two containers of wet ice, one container of dry ice, and a benchtop liquid nitrogen Dewar flask.</li>
	<li>Thaw phenylmethylsulfonyl fluoride (PMSF), pepstatin A, aprotinin, and leupeptin stock solutions on ice (see Table of Materials). Prepare the necessary buffers (Table 1). 
	NOTE: Sucrose solutions can be prepared in advance and stored at 4 °C. However, protease inhibitors (thawed stocks and tablets) must be added fresh to all the buffers at the start of the experiment due to the instability of these reagents in aqueous solutions. Further, all the buffers must be prepared with detergent-free glassware and detergent-free water to enable the collection of intact synaptosomes.</li>
	<li>Chill all the buffers and glass Dounce homogenizers (see Table of Materials) on ice. Set the centrifuges to 4 °C and chill the rotors to 4 °C.</li>
	<li>Add 14 mL of Buffer A (Table 1) to a Dounce homogenizer on ice.</li>
</ol>
Table 1: Composition of the subcellular fractionation buffers. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64574/64574_Table%201_revised.xlsx" target="_blank">Please click here to download this Table.</a>
<img alt="Synaptosome preparation diagram; experimental brain excision, lysis, and synaptic vesicle isolation." class="xfigimg" src="/files/ftp_upload/64574/64574fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Overview of the subcellular fractionation protocol. Summary schematic of the subcellular fractionation steps and collected samples. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64574/64574fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Mouse brain excision
<img alt="Rodent skull anatomy diagram; identifies sutures and bones for comparative anatomical study." class="xfigimg" src="/files/ftp_upload/64574/64574fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Craniofacial anatomy. (A) Dorsal view of a mouse skull with relevant cranial structures indicated. (B) Left lateral view of a mouse skull and brain with relevant cranial structures and anatomical directions indicated. The dashed lines represent the locations where incisions should be made. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64574/64574fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Deeply anesthetize each mouse with 100% isofluorane in an anesthesia chamber located in a fume hood or biosafety cabinet using an open drop method34. Sacrifice each mouse by cervical spine dislocation followed swiftly by decapitation. Alternate between genotypes or experimental groups for each sacrifice and dissection12. Obtain tail biopsies after euthanasia by excising 2 mm of the distal tail tip with fine scissors. Store the tissue for genotyping.</li>
	<li>Spray the decapitated head with 70% ethanol to prevent hair from adhering to the tissue and surgical instruments during dissection.</li>
	<li>Insert fine scissors under the skin at the decapitation incision to a pericranial depth and make a midsagittal incision up to the internasal suture (Figure 3A) to retract the scalp from the skull.</li>
	<li>Working from the occipital area toward each temporal aspect, trim the fascia and muscle to expose the external surface of the skull beyond each external acoustic meatus (Figure 3B).</li>
	<li>Secure the scalp and rostral aspect of the skull with the non-dominant hand. With the other, insert fine scissors 2 mm into the caudal side of the foramen magnum, where the spinal cord is visible exiting. Make a midline incision until the scissors reach the internal surface of the intraparietal bone (Figure 3; dashed lines). 
	NOTE: During the initial incision, the scissors must be parallel to the spinal cord with pressure applied toward the internal surface of the skull to prevent damage to the brainstem and cerebellum.</li>
	<li>Change the angle of the scissors so the blades run parallel with the dorsal surface of the skull. Continue advancing the midsagittal incision rostrally through the parietal and frontal bones, using the sagittal and interfrontal sutures as a guide. Use constant upward pressure to avoid damage to the cortex. Terminate the incision just beyond the internasal suture (Figure 3A).</li>
	<li>Make a small perpendicular incision (~3 mm) to the nasal bone, rostral to the internasal suture, by placing the scissors perpendicular to the skull with each blade positioned at a nasal-premaxillary suture and making one even cut (Figure 3; dashed lines). 
	NOTE: This step will increase the ease of retracting the skull and will be critical for collecting the olfactory bulb if this area is of interest.</li>
	<li>While securing the rostral aspect, use one side of a pair of textured forceps to gently lift the skull up from the brain, then laterally and ventrally. Repeat along the midline as needed, then for the other hemisphere until the entire brain surface is exposed.</li>
	<li>Using curved forceps or a fine spatula, gently lift the rostral side of the brain. Cut the optic and cranial nerves to complete the excision from the skull.</li>
	<li>For each condition, collect five to eight mouse brains together into the chilled glass Dounce homogenizer containing 14 mL of Buffer A (Table 1).</li>
</ol>
3. Synaptosome preparation
NOTE: The schematics of this procedure are shown in Figure 4.
<img alt="Synaptosome preparation diagram; centrifugation steps; biochemical isolation; S1, S2, P2 stages." class="xfigimg" src="/files/ftp_upload/64574/64574fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Synaptosome preparation. Schematic of step 3, the generation of synaptosomes (P2'). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64574/64574fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Homogenize the brains using a glass Dounce homogenizer in 12 up-down passes at 500 rpm (total). Pause briefly at each downstroke to ensure thorough homogenization of the tissue. Homogenize preferentially in an ice bath to avoid warming and protein denaturation. Take 5 µL aliquots for protein concentration determination by the bicinchoninic acid assay (BCA, see Table of Materials). Take 100 µL of whole brain lysate aliquots for western blot (WB). For this and all subsequent samples (Figure 2), take two aliquots for BCA and two aliquots for WB. Flash-freeze all the collected aliquots in liquid nitrogen and store them at −80 °C.</li>
	<li>Spin the total brain homogenate in a high-speed round bottom centrifuge tube (14 mL) (see Table of Materials) at 800 x g for 10 min at 4 °C to obtain the supernatant (S1). Transfer S1 to a new centrifuge tube, leaving the pellet behind (P1), which contains intact cells and nuclei. Avoid pipetting up the fluffy, white, loose, superficial pellet. Take 2 x 5 µL of S1 for BCA and 2 x 100 µL of S1 for WB.</li>
	<li>Spin S1 at 9,000 x g for 15 min at 4 °C to obtain the synaptosomal supernatant (S2) and crude synaptosome pellet (P2). Take 2 x 10 µL of S2 for BCA and 2 x 500 µL of S2 for WB. Discard the supernatant after obtaining aliquots and proceed to the next step with the pellet.</li>
	<li>Resuspend P2 in 3 mL of ice-cold Buffer A with protease inhibitors and centrifuge at 9,000 x g for 15 min at 4 °C to obtain the supernatant (S2') and washed synaptosomes (P2'). Discard the supernatant and keep the pellet.</li>
	<li>Resuspend P2' in 3 mL of Buffer A. Avoid resuspending the dark red portion at the bottom of the pellet, which mainly contains mitochondria. Take 2 x 20 µL of P2' for BCA and 2 x 100 µL of P2' for WB. 
	NOTE: This can be achieved by gently pipette-mixing the edges and surface of the pellet to resuspend the white washed synaptosomes while directing the pipette tip away from the red center of the pellet.</li>
</ol>
4. Hypotonic lysis
NOTE: The schematics of this procedure are shown in Figure 5.
<img alt="Hypotonic lysis process; centrifugation; tube setup; stepwise cell disruption; lab procedure." class="xfigimg" src="/files/ftp_upload/64574/64574fig05.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: Hypotonic lysis. Schematic of step 4, the hypotonic lysis of synaptosomes to generate the lysis supernatant (LS1) and synaptosomal membrane fractions (LP1). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64574/64574fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>For the hypotonic lysis of washed synaptosomes, add 9 volumes of chilled Buffer B (Table 1) to resuspended P2' (~27 mL). Homogenize the synaptosomes in a glass Dounce homogenizer (three up-down passes at 500 rpm).</li>
	<li>Transfer the samples to 50 mL capped conical centrifuge tubes. Rotate them on a tube revolver in a 4 °C cold room for 15 min.</li>
	<li>Centrifuge lysed P2' at 25,000 x g for 20 min at 4 °C to obtain the lysis supernatant (LS1) and the lysis pellet containing synaptosomal membranes (LP1). Take 2 x 50 µL of LS1 for BCA and 2 x 400 µL of LS1 for WB. Transfer LS1 into a capped centrifuge tube for ultracentrifugation (see Table of Materials).</li>
</ol>
5. Synaptic vesicle isolation
NOTE: The schematics of this procedure are shown in Figure 6.
<img alt="Synaptic vesicle and plasma membrane isolation diagram; centrifugation, shear/resuspend steps." class="xfigimg" src="/files/ftp_upload/64574/64574fig06.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6: Synaptic vesicle isolation and synaptic plasma membrane isolation. (A) Schematic of step 5, the isolation of synaptic cytosol (LS2) and synaptic vesicle (LP2) fractions, and (B) step 6, the generation of myelin (MF), synaptic plasma membrane (SPM), and mitochondrial (Mito.) fractions following the ultracentrifugation of sucrose gradients. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64574/64574fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Centrifuge LS1 in a fixed angle ultracentrifuge rotor (see Table of Materials) at 100,000 x g for 60 min at 4 °C to obtain synaptic cytosol supernatant (LS2) and synaptic vesicle pellet (LP2). LP2 will be small, translucent, and strongly adhered to the side of the centrifuge tube.</li>
	<li>Resuspend LP2 in 500 µL of Buffer A. Using a 23 G needle and a 1 mL syringe, shear LP2 with gentle trituration. Take 2 x 10 µL of LP2 for BCA and 2 x 250 µL of LP2 for WB.</li>
	<li>Transfer LS2 (~30 mL) to centrifugal filter units with a 10 kDa cutoff (see Table of Materials). 
	NOTE: If proteins smaller than 10 kDa are of interest, 4 kDa cutoff centrifugal filter units are available but will result in longer spin times.</li>
	<li>Concentrate LS2 to approximately 0.5 mL by spinning at 5000 x g for up to 1 h at 4 °C. Take 2 x 10 µL of concentrated LS2 for BCA and 2 x 250 µL of concentrated LS2 for WB. After starting the spin, proceed directly to step 6.1.</li>
</ol>
6. Synaptic plasma membrane isolation
<ol>
	<li>Resuspend LP1 (step 4.3) in 1 mL of Buffer B (Table 1). Take 2 x 10 µL of LP1 for BCA and 2 x 50 µL of LP1 for WB. Adjust the remaining LP1 to a final volume of 7.5 mL and a final sucrose concentration of 1.1 M with Buffer B and Buffer C (Table 1).</li>
	<li>Transfer 7.5 mL of resuspended LP1 into a 14 mL ultracentrifuge tube (see Table of Materials). Carefully overlay LP1 with 3.75 mL of Buffer D (Table 1), and then overlay with 1.25 mL of Buffer A (or a larger volume to fill just below the top of the centrifuge tube). Avoid pipetting down the side of the tube, which will disrupt the sucrose gradient interfaces. After overlaying each sucrose fraction, mark the top of the solution with a pen. Balance the tubes for ultracentrifugation by weight, not volume, with the dropwise addition of Buffer A to within 10 mg. Centrifuge at 48,000 x g for 2.5 h at 4 °C in a swinging bucket ultracentrifuge rotor (see Table of Materials).</li>
	<li>Acquire images of the intact gradients following ultracentrifugation to document the distinctness of each sucrose interface and the success of fractionation.</li>
	<li>Carefully remove the superficial layer of 320 mM sucrose (Buffer A). Recover the myelin fraction (MF) at the 320 mM/855 mM sucrose interface in an 800 µL volume. Recover the synaptic plasma membrane (SPM) fraction at the 855 mM/1.1 M sucrose interface in a 1,000 µL volume. Pipette each fraction up from the wall of the tube in a circular manner to ensure the complete fraction is collected. Carefully aspirate off the remaining sucrose and recover the mitochondrial pellet (Mito.) by resuspending in 200 µL of Buffer B. Take 2 x 100 µL of MF for BCA and 2 x 10 µL of Mito. for BCA; divide the remainder of MF and Mito. samples in half for WB.</li>
	<li>Dilute the SPM fraction with 2 volumes of Buffer B (~2 mL), and then centrifuge in a fixed angle rotor in a 3.5 mL centrifuge tube (see Table of Materials) at 25,000 x g for 20 min at 4 °C. Discard the supernatant and resuspend the SPM pellet in Buffer A for a final volume of 250 µL. Take 2 x 5 µL of SPM for BCA and divide the remaining SPM in half for WB.</li>
	<li>Perform a BCA to determine the protein concentration of each sample, accounting for variable aliquot volume. 
	NOTE: For WB analysis, the suggested working protein concentration for all subcellular fractions is 2 µg/µL (or as high as achievable for LS1 and MF).</li>
</ol>

The authors have nothing to disclose.

In their seminal studies, Whittaker and colleagues used four morphological criteria to identify synaptosomes: (1) the structures have a sealed plasma membrane; (2) the structures contain SVs resembling those in nerve terminals and varicosities in situ in size and number; (3) the structures possess one or more small mitochondria; and (4) the presynaptic membrane is frequently adhered to&#160;a post-synaptic component11,12,13.&#160;Though the first two criteria generally apply to every isolation method, in the most recent protocols described in this article, not all resulting synaptosomes will have mitochondria and attached post-synaptic terminals. Approximately 60% of the synaptosomes will have mitochondria, and only up to 15% are estimated to have attached post-synaptic terminals37. If post-synaptic components are of particular interest, the use of an isotonic Krebs-like homogenization buffer and pressure filtration for enrichment are known to yield high concentrations of synaptosomes with post-synaptic terminals (also termed synaptoneurosomes)22,38.
The method of sacrificing the animal can impact the quality of synaptosomes and synaptic subfractions. Adult animals sacrificed using a euthanasia method that does not require anesthesia will result in the best fraction quality. Further, the brains should be freshly dissected, not frozen, and homogenized using a 1:10 ratio of homogenization buffer (weight/volume) for the most viable synaptic fractions22. The brain has a heterogeneous population of synapses that can be differentiated by the type of neurotransmitters they carry. Synaptosome formation is generally unaffected by synapse type or neurotransmitter content13. An exception is mossy fibers in the cerebellum, which are known to be disrupted in optimal conditions for obtaining synaptosomes from the rest of the brain39,40. Thus, removal of the cerebellum prior to brain homogenization is recommended if the exclusion of this region does not affect the experimental goal. If interested in isolating synaptosomes of a particular neurotransmitter character, areas of the brain that are enriched for neurons containing the neurotransmitter of interest can first be isolated. However, this approach will impose limitations on the final fraction yield, depending on the size of the region of interest (the age of animals is also, therefore, a consideration). There are immunochemical methods for the isolation of neurotransmitter-specific synaptosomes, but the viability and yield will be significantly compromised22. If assessing synaptosome metabolic viability is important, the measurement of neurotransmitter release41,42 or certain enzymatic assays43 can be employed.
Common contaminants in synaptosome preparations include microsomes, free mitochondria, SVs, and neuronal and glial membranes. Contamination can be reduced by increasing the number of washes at the P1 and P2 fractions22 and avoiding resuspension of the red mitochondrial pellet in subsequent steps. In experiments where metabolic viability and time are crucial, reducing the number of washes and using Ficoll or Percoll gradients over sucrose gradients will be helpful44,45,46. These methods also reduce contamination significantly. Whittaker&#39;s original protocol yielded high-quality SVs. Further optimization by Nagy et al.23, included in this method, produces SVs with remarkable homogeneity and purity without compromising significantly on yield36. If specific SV subtypes are of interest, such as glutamatergic (VGLUT-1-containing) or GABAergic (VGAT-1-containing) SVs, immunoisolation using specific antibodies can be performed47,48. Alternative methods are also available for isolating CCVs from synaptosomes, which, due to differential density, may not be present at the same interface as SVs obtained with this method20,49,50.
Overall, the present protocol to isolate synaptic components can be further optimized to obtain fractions with improved homogeneity and viability based on the quality and quantity of the source brain tissue and the experimental goals. For further troubleshooting details, one should refer to book chapters by Dunkley and Robinson22 and Ganzella et al.36.

Synapses are the basic computational units of the brain through which neurons communicate and exert diverse and exquisitely complex functions. Synapses are, thus, fundamental to the health of the brain1; synaptic dysfunction is implicated as a source or result of many disorders2. Synapses are constituted by pre- and post-synaptic terminals, extensions of two different neurons that are closely apposed and separated by a synaptic cleft traversed by synaptic adhesion molecules. Information flows from the pre- to post-synaptic compartment in the form of chemical messengers called neurotransmitters1. The molecular processes involved in neurotransmission are active areas of research3,4,5. Understanding the pathogenic processes within synaptic terminals and the response of synapses to pathology in other neuronal sub-compartments are crucial steps to addressing disorders of the brain1,2. Several methodological advancements, predominantly applied to murine models, have advanced this pursuit6. The isolation of synaptic fractions by differential centrifugation is one such paradigm-shifting method that has enabled the detailed evaluation of synaptic processes in health and disease.
The adult human brain consists of 80-90 billion neurons7,8. Among murine species, the rat brain contains approximately ~200 million neurons, while mice have ~70 million9,10. Each neuron forms thousands of specific synaptic connections with a network of highly polarized neurons intermingled with glial cells and dense vasculature. In such complex and heterogeneous tissue, it was once unthinkable to isolate and study synapses as an independent system. In the 1960s, Victor Whittaker, Catherine Hebb, and others made this possible by isolating intact synaptic terminals using subcellular fractionation11,12,13,14. In an attempt to isolate synaptic vesicles (SVs), they homogenized brains through liquid shear force in iso-osmotic (0.32 M) sucrose followed by ultracentrifugation. They obtained pinched-off, plasma membrane-enclosed, intact nerve terminals or varicosities, which they called nerve-ending particles (NEPs)11,13. As the structural and functional characteristics of the synapse were preserved in these structures, NEPs were later termed &#34;synaptosomes&#34; for congruence with other subcellular organelles13,15. It is worth noting that the work of Eduardo de Robertis and colleagues, who coined the term &#34;synaptic vesicle&#34;, overlapped with that of Whittaker and colleagues and contributed to the validation of &#34;synaptosome&#34; isolation and characterization16,17,18.
Synaptosomes are physiologically active structures that contain all the cellular and molecular properties required for the storage, release, and reuptake of neurotransmitters13,18. The preservation of key synaptic characteristics in vitro and freedom from non-synaptic components also contribute to the utility of this isolation method. Synaptosomes have contributed immensely to the understanding of the chemical and physiological properties of neurotransmission and are now being used to study synaptic molecular processes and their alterations in disease19,20,21,22,23. Synaptosomes are also the initial source material for isolating synaptic components such as SVs, clathrin-coated vesicles (CCVs), synaptic cytosol, synaptic plasma membrane, synaptic mitochondria, synaptic adhesion molecules, and other components of interest, which can facilitate the understanding of the molecular mechanisms of synaptic function18,19,20,24,25,26,27,28. These sub-synaptic components can be obtained by the osmotic lysis of synaptosomes and sucrose density gradient ultracentrifugation15,29. Although the original subcellular fractionation method by Whittaker&#39;s research group is known to be efficient in isolating quality synaptosomes and SVs13,30, recent optimizations enhance the purity of the subcellular fractions22,23,31,32. This article provides a highly detailed and accessible version of a classic protocol for the subcellular fractionation of murine brain tissue to isolate synaptosomes, SVs, and other sub-synaptic components.

<table><tbody><tr><td>1 mL TB Syringe</td><td>BD</td><td>309649</td><td></td></tr><tr><td>1.5 mL Eppendorf Tubes</td><td>USA Scientific</td><td>1415-2500</td><td></td></tr><tr><td>14 mL, Open-Top Thinwall Ultra-Clear Tube</td><td>Beckman Coulter</td><td>344060</td><td>Compatible with SW 40 Ti</td></tr><tr><td>23 Gauge Precision Glide Hypodermic Needle</td><td>BD</td><td>305145</td><td></td></tr><tr><td>26.3 mL, Polycarbonate Bottle with Cap Assembly</td><td>Beckman Coulter</td><td>355618</td><td>Compatible with Ti70</td></tr><tr><td>3.5 mL, Open-Top Thickwall Polypropylene Tube</td><td>Beckman Coulter</td><td>349623</td><td>Compatible with TLA-100.3</td></tr><tr><td>50 mL Falcon Tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Unit&amp;nbsp;</td><td>Millipore Sigma</td><td>UFC901024</td><td></td></tr><tr><td>Aprotinin</td><td>Sigma-Aldrich</td><td>A6279</td><td>1 mg/mL in diH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Avanti J-26 XP Centrifuge</td><td>Beckman Coulter</td><td>B22984</td><td>&amp;lt;26,000 rpm</td></tr><tr><td>Benchtop HDPE Dewar Flask</td><td>Thermo Scientific</td><td>5028U19</td><td></td></tr><tr><td>C57BL/6J Mice</td><td>The Jackson Labs</td><td>000664</td><td></td></tr><tr><td>Centrifuge 5810R</td><td>Eppendorf</td><td>EP022628168</td><td>&amp;lt;14,000 rpm</td></tr><tr><td>complete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets</td><td>Roche</td><td>11873580001</td><td>Add 1 tablet per 50 mL of solution</td></tr><tr><td>Curved Forceps</td><td>Fine Science Tools</td><td>11273-20</td><td></td></tr><tr><td>Fine Surgical Scissors</td><td>Fine Science Tools</td><td>8r</td><td></td></tr><tr><td>Glas-Col Tissue Homogenizing System</td><td>Cole-Parmer</td><td>UX-04369-15</td><td></td></tr><tr><td>Graefe Forceps</td><td>Fine Science Tools</td><td>11650-10</td><td></td></tr><tr><td>High-Speed Polycarbonate Round Bottom Centrifuge Tubes</td><td>ThermoFisher</td><td>3117-0500</td><td>Compatible with JA20</td></tr><tr><td>Isofluorane</td><td>Henry Schein Animal Health</td><td>NDC 11695-6776-2</td><td></td></tr><tr><td>JA-20 Rotor</td><td>Beckman Coulter</td><td>334831</td><td></td></tr><tr><td>Leupeptin</td><td>American Bio</td><td>AB01108</td><td>1 mg/mL in diH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>N-[2-Hydroxyethyl] piperazine-N&amp;rsquo;-[2-ethanesulfonic acid] (HEPES)</td><td>American Bio</td><td>AB00892</td><td></td></tr><tr><td>Optima L-80 XP Ultracentrifuge</td><td>Beckman Coulter</td><td></td><td>&amp;lt;100,000 rpm</td></tr><tr><td>Optima TLX Ultracentrifuge</td><td>Beckman Coulter</td><td></td><td>&amp;lt;120,000 rpm</td></tr><tr><td>Pepstatin A</td><td>Thermo Scientific</td><td>78436</td><td>1 mg/mL in DMSO</td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>American Bio</td><td>AB01620</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23335</td><td>For determination of protein concentration</td></tr><tr><td>Pipette Tips</td><td></td><td></td><td></td></tr><tr><td>Serological Pipettes</td><td></td><td></td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools</td><td>14002-12</td><td></td></tr><tr><td>SW 40 Ti Swinging-Bucket Rotor</td><td>Beckman Coulter</td><td>331301</td><td></td></tr><tr><td>Teflon-Coated Pestle and Mortar Tissue Grinder</td><td>Thomas Scientific</td><td>3431D94</td><td></td></tr><tr><td>Ti70 Rotor</td><td>Beckman Coulter</td><td>337922</td><td></td></tr><tr><td>TLA-100.3 Rotor</td><td>Beckman Coulter</td><td>349490</td><td></td></tr><tr><td>Tube Revolver</td><td>Dot Scientific</td><td>DTR-02VS</td><td></td></tr></tbody></table>

subcellular fractionation for the isolation of synaptic components from the murine brain

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological disorders. The characterization of synaptic sub-compartments by biochemical isolation is, therefore, a powerful method to elucidate the molecular bases of synaptic processes, both in health and disease. This protocol describes the isolation of synaptic terminals and synaptic sub-compartments from mouse brains by subcellular fractionation. First, sealed synaptic terminal structures, known as synaptosomes, are isolated following brain tissue homogenization. Synaptosomes are neuronal pre- and post-synaptic compartments with pinched-off and sealed membranes. These structures retain a metabolically active state and are valuable for studying synaptic structure and function. The synaptosomes are then subjected to hypotonic lysis and ultracentrifugation to obtain synaptic sub-compartments enriched for synaptic vesicles, synaptic cytosol, and synaptic plasma membrane. Fraction purity is confirmed by electron microscopy and biochemical enrichment analysis for proteins specific to sub-synaptic compartments. The presented method is a straightforward and valuable tool for studying the structural and functional characteristics of the synapse and the molecular etiology of various brain disorders.

The presented method results in 11 brain subcellular fractions that can be subjected to further purification and various forms of downstream analysis35,36. The gold standard method to assess the quality of synaptosomes, SVs23, and other components is electron microscopy (EM) (Figure 7). Quantitative immunoblotting for proteins that are present in specific subcellular fractions can also be performed to assess markers of fraction purity (Figure 8). For example, immunoblot analysis of fractions reveals the enrichment of N-cadherin (CDH2, UniProt name) in the synaptic plasma membrane fraction (SPM), α-synuclein (SYUA) in the synaptic cytosol (LS2), synaptophysin (SYPH) in the synaptic vesicle fraction (LP2), and myelin basic protein (MBP) in the myelin fraction (MF) when compared to protein levels in the initial whole brain homogenate (Total) (Figure 8). Once fraction purity has been established (for example, note the absence of CDH2 in the LS2 fraction or the many-fold increase in SYPH in the LP2 fraction), quantitative immunoblotting can be used to determine the localization of proteins of interest or query differences in protein distribution between genotypes or treatments. Understanding the subcellular localization of synaptic proteins can enable the dissection of previously undescribed protein functions. Further, this method may elucidate trafficking defects or synaptic dysfunction in disease states, especially when paired with functional assays. For example, our team has used this method to identify a pool of enzymatically active palmitoyl protein thioesterase 1 that is enriched in the synaptic cytosol19.
<img alt="Cell organelle structure; electron microscopy images A, B, C; cellular component analysis." class="xfigimg" src="/files/ftp_upload/64574/64574fig07.jpg" data-altupdatedat="1747911167000" data-alt="Figure 7"> 
Figure 7: Electron microscopy (EM) of synaptosomes. (A) Representative EM image of a synaptosome containing synaptic vesicles (arrow). (B) Representative EM image of a synaptosome with both pre- (arrow) and post-synaptic components (double arrow). (C) Representative EM image of a synaptosome containing synaptic vesicles and a mitochondrion (arrow) (scale bars = 100 nm). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64574/64574fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Western blot analysis and bar chart of protein markers in cell fractions, showing marker enrichment." class="xfigimg" src="/files/ftp_upload/64574/64574fig08.jpg" data-altupdatedat="1747911167000" data-alt="Figure 8"> 
Figure 8: Immunoblot analysis of subcellular fractions. (A) Markers of subcellular fraction purity (indicated with UniProt nomenclature) are appropriately localized compared to the whole brain homogenate (total): N-cadherin (CDH2) in the synaptic plasma membrane fraction (SPM), synaptophysin 1 (SYPH) and synaptojanin 1 (SYNJ1) in the synaptic vesicle enriched fraction (LP2), α-synuclein (SYUA) in the synaptic cytosol (LS2), and myelin basic protein (MBP) in the myelin fraction (MF). (B) Immunoblot quantification analysis reveals the enrichment (fold-change from total) of fraction purity markers. Data are represented as mean ± standard deviation on a log10 scale. The dotted line indicates a 1.5-fold change (y = 0.176) (n = 3 replicate experiments with 8 wild-type mice; age = 2 months; n = 4-5 blots for SYPH, SYUA, MBP, with n = 3 plotted values previously published by Gorenberg et al.19; n = 5 for SYNJ1; n = 1 for CDH2). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64574/64574fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain at JoVE.com

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

This protocol presents a robust, detailed method to obtain highly pure synaptosomes, synaptic vesicles, and other synaptic fractions from the mouse brain. This method enables the evaluation of synaptic processes, including the biochemical analysis of protein localization and function with compartmental resolution.

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological ...

subcellular-fractionation-for-isolation-synaptic-components-from

Nanyang Technological University

Research

JoVE Journal

Neuroscience

5.9K Views.  Yale University. Synapsen zijn de basis computationele eenheden van de hersenen. De gepresenteerde methode is een eenvoudig en waardevol hulpmiddel om de synapsen en hun moleculaire processen te bestuderen en ook om te begrijpen hoe ze kunnen worden veranderd bij verschillende hersenaandoeningen. Deze techniek omvat een fractionering van synaptosoomstructuren geïsoleerd uit de hersenen.Dit stelt de onderzoeker in staat om effecten vast te leggen die mogelijk worden gecompartimenteerd op de synaps. De ...

Video: Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Biochemistry

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS induces generalized tonic-clonic seizures with low mortality and neuronal death and is a widely-used model to screen anti-epileptic drugs. Here, we describe an ECS induction method in which a brief 55-mA current is delivered for 0.5 s to male rats 200 - 250 g in weight via ear-clip electrodes. Such bilateral stimulation produced stage 4 - 5 clonic seizures that lasted about 10 s. After the cessation of acute or chronic ECS, most rats recovered to be behaviorally indistinguishable from sham &#34;no seizure&#34; rats. Because ECS globally elevates brain activity, it has also been used to examine activity-dependent alterations of synaptic proteins and their effects on synaptic strength using multiple methods. In particular, subcellular fractionation of the postsynaptic density (PSD) in combination with Western blotting allows for the quantitative determination of the abundance of synaptic proteins at this specialized synaptic structure. In contrast to a previous fractionation method that requires large amount of rodent brains, we describe here a small-scale fractionation method to isolate the PSD from the hippocampi of a single rat, without sucrose gradient centrifugation. Using this method, we show that the isolated PSD fraction contains postsynaptic membrane proteins, including PSD95, GluN2B, and GluA2. Presynaptic marker synaptophysin and soluble cytoplasmic protein &#945;-tubulin were excluded from the PSD fraction, demonstrating successful PSD isolation. Furthermore, chronic ECS decreased GluN2B expression in the PSD, indicating that our small-scale PSD fractionation method can be applied to detect the changes in hippocampal PSD proteins from a single rat after genetic, pharmacological, or mechanical treatments.

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy for severe depression. ECS globally stimulates activity in the hippocampus, leading to synaptogenesis and synaptic plasticity. Here, we describe methods for ECS induction in rats and for subcellular fractionation of their hippocampi to examine seizure-induced changes in synaptic proteins.

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in preparation for detailed downstream analysis such as flow cytometry or single-cell sequencing. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination thereof. The delicate nature of neuronal cells can complicate efforts to obtain the highly viable, true single-cell suspension with minimal cellular debris that is required for single-cell analysis. The data demonstrate that this combination of automated mechanical dissociation and enzymatic digestion consistently yields a highly viable (&gt;90%) single-cell suspension, overcoming the aforementioned difficulties. While a few of the steps require manual dexterity, these steps lessen sample handling and potential cell loss. This manuscript details each step of the process to equip other laboratories to successfully dissociate small quantities of neural tissue in preparation for downstream analysis.

This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining steps.

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in ...

Mouse Brain Histone Modification Quantification Using Flow Cytometry

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

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 ...

Mononuclear Cell Isolation from Mouse Brain: A Density Gradient Centrifugation Technique to Isolate Brain Resident Mononuclear Cells

Source: Ji, Z. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/61050/flow-cytometric-analysis-lymphocyte-infiltration-central-nervous">Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis.</a> J. Vis. Exp. (2020)
In this video, we perform the isolation of mononuclear cells from a mouse&#8217;s brain using density gradient centrifugation. The isolated mononuclear cell suspension can be used for further downstream analysis.

Source: Ji, Z. et al. Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis. J. ...

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

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