This work was supported by Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-1-015), the National Key Research and Development Project of China (2019YFA0801500), the National Natural Science Foundation of China (NSFC: 81970243, 81770308), Beijing Natural Science Foundation (7172183, 7182140).

All experiments were conducted in accordance with the Guide for the Use and Care of Laboratory Animals. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC), Fuwai Hospital, Chinese Academy of Medical Sciences. Aseptic technique is required throughout the procedure to prevent contamination of the surgical site.
1. Apical resection operation in neonatal 1-day-old Cx3cr1 GFP/+ mouse (C57BL/6 background)
<ol>
	<li>Anesthesia
	<ol>
		<li>Take all the mouse pups out from the cages and put them in a clean dry box.</li>
		<li>Embed each mouse in the ice for about 2-3 minutes. Ensure that there is a thin barrier such as latex or gauze between the ice and the mouse pup to prevent&#160;frostbite.&#160;</li>
		<li>Identify sufficient anesthesia by observing for the following signs: pale skin, lack of limb movement, and lack of pedal reflex.</li>
	</ol>
	</li>
	<li>Thoracotomy
	<ol>
		<li>Precool the bronze operating platform overnight at -20 &#176;C to help maintain anesthesia.</li>
		<li>Take the anesthetized mouse out of the ice box and put it on a bronze operating platform. Anchor the mouse on the operating platform in a supine position using medical adhesive tape.</li>
		<li>Put the operating platform with the mouse on it under a stereoscope.</li>
		<li>Disinfect the mouse chest using a prep pad soaked with betadine and 70% alcohol or as consistent with institutional policy.</li>
		<li>Incise the skin with a 1 cm cut at the fourth intercostal area of the chest cavity and then separate the intercostal muscles using microsurgical scissors until the heart is accessed.</li>
		<li>Alternately press the chest and abdomen with the help of two forceps until the heart is exteriorized out of chest without any mechanical damage.</li>
		<li>Set the ribs below and above the small incision as a natural fixation to immobilize the heart.</li>
	</ol>
	</li>
	<li>Ventricular apex resection
	<ol>
		<li>Locate the apex of the left ventricle. Cut a 1 mm diameter of ventricular apex tissue using iridectomy scissors under a stereoscope.</li>
		<li>Confirm that the left ventricular chamber is exposed, and begin oozing.</li>
		<li>Gently press the heart back to chest cavity using a cotton swab.</li>
		<li>Suture the muscles, ribs, and skin using 8-0 Prolene sutures.</li>
		<li>Clean the mouse thoroughly after the operation.</li>
	</ol>
	</li>
	<li>Post-operation care
	<ol>
		<li>Transfer the mouse from the operating platform to a 37 &#176;C heating blanket to warm up the body immediately after the operation.</li>
		<li>Confirm anabiosis of the mouse by observing for the following signs: spontaneous respiration restoration, skin color change from pale to pink, and movement of limbs.</li>
		<li>Take the operated mouse back to its mother once it has recovered.</li>
		<li>Mingle the operated mouse with its mother&#39;s nesting materials if necessary. 
		&#8203;NOTE: It is important to remove all the 1-day-old pups from the mother at once and then return them all at once after all the pups are recovered.</li>
	</ol>
	</li>
</ol>
2. Preparation of the neonatal cardiac macrophage suspension
NOTE: All these experimental procedures should be carried out in a dark place and under sterile conditions.
<ol>
	<li>Harvest the heart of a neonatal Cx3cr1GFP/+ mouse 1-day after apical resection
	<ol>
		<li>Euthanize the Cx3cr1GFP/+ mouse one day after the apical resection. Use an excess of carbon dioxide and decapitate the mouse sequentially to apply euthanasia.</li>
		<li>Take the heart out of the chest and immerse it in a 10 cm dish with PBS.</li>
		<li>Cut the vessels and remaining connective tissue away from the ventricles. Cut the auricular appendix and outflow tract away from the heart.</li>
		<li>After the heart stops beating, cut the heart into 1-2 mm3 pieces in PBS buffer using microsurgical scissors (Figure 2A).</li>
	</ol>
	</li>
	<li>Neonatal mouse heart dissociation
	<ol>
		<li>Transfer the harvested heart tissue to a tube containing 2.5 mL of preheated enzyme mix (Table of Materials), and tightly close the tube.</li>
		<li>Invert the tube and place it with the cap down (Figure 2B).</li>
		<li>Run the neonatal heart dissociation program (Table of Materials).</li>
		<li>Detach the tube from the dissociator after the program is completed.</li>
		<li>Add 7.5 mL of 1x DMEM with 10% FBS to the tube.</li>
		<li>Filter and transfer the suspension to a 15 mL centrifuge tube (Figure 2C). Centrifuge the cell suspension at 300 x g for 5 minutes.</li>
		<li>Resuspend the cell pellet in 1 mL of blood cell lysis solution and incubate for 2 minutes at room temperature.</li>
		<li>Add 5-10 mL of PBS buffer to the suspension and centrifuge at 300 x g for 5 minutes.</li>
		<li>Resuspend the cell pellet in 1 mL of DMEM with 10% FBS.</li>
	</ol>
	</li>
	<li>Neonatal cardiac macrophage isolation
	<ol>
		<li>Sort GFP+ neonatal cardiac macrophages by FACS. Receive the sorted macrophages in a sterile tube containing 500 &#181;L of DMEM with 10% FBS.</li>
		<li>Count the GFP+ macrophages (Figure 2D). Resuspend the macrophage in DMEM with 10% FBS at the concentration of 1 x 106 macrophages per 200 &#181;L of DMEM for later injection. 
		NOTE: The number of GFP+ macrophages is small in one neonate (approximate 1 x 105); therefore, at least 10 neonates should be used to obtain enough macrophages for each adult mouse injection.</li>
	</ol>
	</li>
</ol>
3. Neonatal cardiac macrophage transplantation
<ol>
	<li>Perform a myocardial infarction operation on an adult (6-8-week-old) male C57BL/6 mouse by ligation of the left anterior coronary artery8.</li>
	<li>Put the operated mouse on a 37 &#176;C heated blanket until it recovers.</li>
	<li>Intravenously inject 200 &#181;L of DMEM with 1 x 106 GFP+ neonatal cardiac macrophages into the infarcted adult mouse after the operation (approximate 6 hours later) via the tail vein.</li>
	<li>Send the mouse back to a clean cage.</li>
</ol>
4. Result evaluation
<ol>
	<li>Injection efficiency validation
	<ol>
		<li>Anesthetize the operated mouse and harvest the heart 7 days after the myocardial infarction operation.</li>
		<li>Immerse the heart in the pre-cooled PBS buffer.</li>
		<li>Fix the heart tissue with 4% polyformaldehyde for 72 hours at room temperature with shaking.</li>
		<li>Dehydrate the heart tissue in dimethylbenzene and ethanol.</li>
		<li>Embed the heart tissue in paraffin and slice it into sections with a thickness of 5 &#181;m.</li>
		<li>Perform the standard immunofluorescence staining protocol1. Use &#945;-actinin to mark cardiomyocytes.</li>
		<li>Detect the GFP+ macrophages in the heart receiving transplantation.</li>
		<li>Perform the standard immunofluorescence staining protocol1. Use &#945;-actinin and pH3 to mark cardiomyocytes and cell proliferation, respectively (Figure 3A).</li>
	</ol>
	</li>
	<li>Cardiac repair evaluation after the transplantation
	<ol>
		<li>Use echocardiography on the operated mouse one month after the operation1.</li>
		<li>Analyze the cardiac function by comparing the left ventricular ejection fraction and fractional shortening in different groups (Figure 3B).</li>
		<li>Anesthetize the operated mouse and harvest the heart.</li>
		<li>Repeat steps 4.1.2-4.1.5.</li>
		<li>Perform a Masson&#39;s staining protocol.</li>
		<li>Analyze the infarcted area after macrophage injection (Figure 3C).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Li, Y., 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=gp130+Controls+Cardiomyocyte+Proliferation+and+Heart+Regeneration.">gp130 Controls Cardiomyocyte Proliferation and Heart Regeneration.</a> Circulation. 142 (10), 967-982 (2020).</li><li>Pei, J., 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=Hydrogen+Sulfide+Promotes+Cardiomyocyte+Proliferation+and+Heart+Regeneration+via+ROS+Scavenging.">Hydrogen Sulfide Promotes Cardiomyocyte Proliferation and Heart Regeneration via ROS Scavenging.</a> Oxidative Medicine and Cellular Longevity. , 1412696 (2020).</li><li>Wang, Y., 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=Mydgf+promotes+Cardiomyocyte+proliferation+and+Neonatal+Heart+regeneration.">Mydgf promotes Cardiomyocyte proliferation and Neonatal Heart regeneration.</a> Theranostics. 10 (20), 9100-9112 (2020).</li><li>Lavine, K. J., 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=Distinct+macrophage+lineages+contribute+to+disparate+patterns+of+cardiac+recovery+and+remodeling+in+the+neonatal+and+adult+heart.">Distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (45), 16029-16034 (2014).</li><li>Aurora, A. 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=Macrophages+are+required+for+neonatal+heart+regeneration.">Macrophages are required for neonatal heart regeneration.</a> Journal of Clinical Investigation. 124 (3), 1382-1392 (2014).</li><li>Han, C., 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=Acute+inflammation+stimulates+a+regenerative+response+in+the+neonatal+mouse+heart.">Acute inflammation stimulates a regenerative response in the neonatal mouse heart.</a> Cell Research. 25 (10), 1137-1151 (2015).</li><li>Li, Y., 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=Transplantation+of+murine+neonatal+cardiac+macrophage+improves+adult+cardiac+repair.">Transplantation of murine neonatal cardiac macrophage improves adult cardiac repair.</a> Cellular &amp; Molecular Immunology. , (2020).</li><li>Yue, Z., 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=PDGFR-&#946;+Signaling+Regulates+Cardiomyocyte+Proliferation+and+Myocardial+Regeneration.">PDGFR-&#946; Signaling Regulates Cardiomyocyte Proliferation and Myocardial Regeneration.</a> Cell Reports. 28 (4), 966-978 (2019).</li><li>Wang, J., 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=A+long+noncoding+RNA+NR_045363+controls+cardiomyocyte+proliferation+and+cardiac+repair.">A long noncoding RNA NR_045363 controls cardiomyocyte proliferation and cardiac repair.</a> Journal of Molecular and Cellular Cardiology. 127, 105-114 (2019).</li><li>Li, Y., 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=Achieving+stable+myocardial+regeneration+after+apical+resection+in+neonatal+mice.">Achieving stable myocardial regeneration after apical resection in neonatal mice.</a> Journal of Cellular and Molecular Medicine. 24 (11), 6500-6504 (2020).</li><li>Vagnozzi, R. J., 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=An+acute+immune+response+underlies+the+benefit+of+cardiac+stem+cell+therapy.">An acute immune response underlies the benefit of cardiac stem cell therapy.</a> Nature. 577 (7790), 405-409 (2020).</li></ol>

No conflicts of interest.

Here, we provide an effective approach to prepare, acquire, and transplant neonatal cardiac macrophages to promote adult mouse heart regeneration.
Apical resection is a simple and effective operation to stimulate heart regeneration. We have optimized the details of apical resection to ensure the maximum survival rate of the animals involved in the operation10. Anesthesia time should not be longer than 3 minutes, which lead to hypothermia-induced death, or shorter than 2 minutes, which would cause excessive bleeding during the operation. The standard apical resection was supposed to amputate about 1.5 mm diameter of the apex tissue. However, the purpose of the operation here was to stimulate abundant macrophage infiltration during heart regeneration process. Resection of less than 1.5 mm diameter was acceptable as it could guarantee a maximum survival rate and effective macrophage recruitment at the same time. Only the skillfulness of the operator influenced the survival rate, and the operated mice could not survive the prolonged operation duration. The operator should complete the whole procedure within 5 minutes.
In our former study, we found that acute inflammation promoted neonatal heart regeneration. Intramyocardial microinjection of immunogenic zymosan A particles into neonatal mouse heart could promote cardiomyocyte proliferation6. Recently, Molkentin et al. claimed that macrophage infiltration that is stimulated by intracardiac injection of zymosan A, cell debris, and freeze/thaw killed cells can promote cardiac repair, confirming that acute inflammation and macrophages are essential in cardiac repair rather than stem cells differentiating into cardiomyocytes11. Sadek et al.5 reported that macrophages could promote neonatal heart regeneration via angiogenesis. Our recent study revealed that neonatal cardiac macrophage injection could promote adult heart regeneration and increase the ability of adult cardiomyocyte proliferation1,7. Neonatal cardiac macrophage transplantation might be a promising strategy to promote adult mouse heart regeneration. Here we present the protocols to help more researchers on the regenerative application development and explore of heart regeneration mechanisms.

Heart regeneration is a promising strategy to recover cardiac function after cardiac injury and to protect against heart failure1,2,3. Following myocardial injury, macrophages infiltrate the injured heart and have been explored as the key factors during neonatal heart regeneration4,5,6. Besides clearing necrotic cellular debris and inducing inflammation, macrophages promote angiogenesis5 and cardiomyocyte proliferation7 after neonatal mice myocardial infarction.
Our previous study illustrates that transplantation of neonatal cardiac macrophages isolated from injured neonatal heart enhances adult heart regeneration7, indicating that neonatal cardiac macrophage transplantation could be a promising strategy to treat cardiac injury. Here we provide the technical details, including isolation of neonatal cardiac macrophages from apical resection-injured neonatal mouse hearts, the transplantation of macrophages in to myocardial-infarcted adult mice, and the estimation of heart regeneration after macrophage graft (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-mouse alpha actinin</td>
			<td>Abcam</td>
			<td>Ab9465</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-phospho-Histone H3</td>
			<td>Millipore</td>
			<td>06-570</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit Aurora B</td>
			<td>Abcam</td>
			<td>Ab239837</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit Ki67</td>
			<td>Abcam</td>
			<td>Ab15580</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Octo Dissociator</td>
			<td>Miltenyi Bio Tech, Teterow, Germany</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse Alexa Fluor 555</td>
			<td>Invitrogen</td>
			<td>A-21137</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Neonatal Heart Dissociation Kit</td>
			<td>Miltenyi Bio Tech, Teterow, Germany</td>
			<td>130-098-373</td>
			<td></td>
		</tr>
	</tbody>
</table>

transplantation of neonatal mouse cardiac macrophages into adult mice

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study reveals that transplantation of neonatal cardiac macrophages recruited by injury promotes adult heart regeneration after myocardial infarction with improvement of cardiac function and cardiomyocyte proliferation. The results indicate that neonatal cardiac macrophage transplantation could be a promising strategy for cardiac injury treatment. Here, we provide the technical details, including the isolation of neonatal cardiac macrophages from apical resection-injured neonatal mouse hearts, the transplantation of macrophages into myocardial-infarcted adult mice, and the estimation of heart regeneration after a macrophage graft.

The protocol described here is summarized in a flow chart (Figure 1). We performed apical resection operation on 1-day-old Cx3cr1GFP/+ mouse. As shown in Figure 2A, the neonatal Cx3cr1GFP/+ mouse was anchored on the operating platform under the stereoscope after anesthesia. We applied pressure alternately on the mouse chest and abdomen with the help of two forceps, which can be set up as a tract to guide the heart popping out of the chest. Any extra mechanical damage to the heart that may affect heart regeneration should be avoided. The heart was immobilized by the surrounding chest tissues, which facilitated the operation on the myocardium. We found that cutting off less than a 1 mm diameter of the resected apex tissue by iridectomy scissors was appropriate when the left ventricular chamber began oozing. Successful induction of apical resection model is necessary for macrophage transplantation8,9.
We sorted the GFP+ macrophages strictly following a neonatal heart dissociation protocol1 (Figure 2).
Neonatal cardiac macrophages were injected into the myocardial infarcted adult mouse heart shortly after the isolation. In order to confirm the efficiency of the macrophage transplantation, we performed immunofluorescence staining at 7 days after the injection. The results showed that GFP+ macrophages could be found in the adult myocardial infarcted mouse heart, indicating the successful transplantation of neonatal cardiac macrophage. We employed co-immunostaining of pH3 with &#945;-actinin. The co-localization was considered to be cardiomyocyte proliferation. The results illustrated that the number of proliferative cardiomyocytes was upregulated in the macrophage-injected group, indicating that adult cardiomyocyte proliferation ability was enhanced after the transplantation (Figure 3).
Adult mouse heart regeneration was evaluated 1 month after the transplantation. We performed echocardiogram on the adult mouse and found that macrophage injection could enhance the cardiac function after myocardial infarction. Masson&#39;s staining was performed, and the results illustrated that infarcted area was remarkably reduced after the neonatal cardiac macrophage transplantation (Figure 3). All these results demonstrated that neonatal cardiac macrophage transplantation promotes adult mouse heart regeneration and cardiomyocyte proliferation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62108/62108fig01.jpg" /> 
Figure 1: Schematic representation of neonatal cardiac macrophage transplantation. <a href="https://www.jove.com/files/ftp_upload/62108/62108fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62108/62108fig02.jpg" /> 
Figure 2: Images of macrophage isolation. A) The hearts are cut into small pieces. B) The mouse hearts are dissociated. C) The suspension is filtered and transferred to a 15 mL centrifuge tube. D) The number of neonatal cardiac macrophages is calculated. <a href="https://www.jove.com/files/ftp_upload/62108/62108fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62108/62108fig03.jpg" /> 
Figure 3: Neonatal cardiac macrophage transplantation promotes adult heart regeneration. A) (Left) Immunofluorescence images show the proliferative cardiomyocytes (arrow, pH3 green, &#945;-actinin red). (Right) Statistical analysis show that cardiomyocyte proliferation increases after macrophage transplantation. B) (Left) Echocardiogram images show the cardiac function in adult mouse 1-month after myocardial infarction. (Right) Statistical analysis show that cardiac function is enhanced after macrophage transplantation. C) Masson&#39;s staining shows the infarcted area in adult mouse 1-month after myocardial infarction. (Right) Statistical analysis show that infarcted area is reduced after M&#981;, macrophage transplantation. <a href="https://www.jove.com/files/ftp_upload/62108/62108fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice at JoVE.com

Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice

We provide a protocol for neonatal cardiac macrophage separation and transplantation into an adult mouse heart, which could be a promising way to promote cardiac repair.

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study ...

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2.7K Views.  Peking Union Medical College. We provide a protocol for neonatal cardiac macrophage separation and transplantation into an adult mouse heart, which could be a promising way to promote cardiac repair.

Video: Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice

Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of recordings are conducted on dissociated DRG neurons, which is a standard preparation for most laboratories. Neuronal properties, however, can be altered by axonal injury resulting from enzyme digestion used in acquiring dissociated neurons. Further, dissociated neuron preparations cannot fully represent the microenvironment of the DRG since loss of contact with satellite glial cells that surround the primary sensory neurons is an unavoidable consequence of this method. To overcome the limitations in using conventional dissociated DRG neurons for patch clamp recordings, in this report we describe a method to prepare intact DRGs and conduct patch clamp recordings on individual primary sensory neurons ex vivo. This approach permits the fast and straightforward preparation of intact DRGs, mimicking in vivo conditions by keeping DRG neurons associated with their surrounding satellite glial cells and basement membrane. Furthermore, the method avoids axonal injury from manipulation and enzyme digestion such as when dissociating DRGs. This ex vivo preparation can additionally be used to study the interaction between primary sensory neurons and satellite glial cells.

This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

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A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

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Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

Studying Protein Import into Chloroplasts Using Protoplasts

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many secondary metabolites and lipids. Chloroplasts require a large number of proteins for these various physiological processes. Over 95% of chloroplast proteins are nucleus-encoded and imported into chloroplasts from the cytosol after translation on cytosolic ribosomes. Thus, the proper import or targeting of these nucleus-encoded chloroplast proteins to chloroplasts is essential for the proper functioning of chloroplasts as well as the plant cell. Nucleus-encoded chloroplast proteins contain signal sequences for specific targeting to chloroplasts. Molecular machinery localized to the chloroplast or cytosol recognize these signals and carry out the import process. To investigate the mechanisms of protein import or targeting to chloroplasts in vivo, we developed a rapid, efficient protoplast-based method to analyze protein import into chloroplasts of Arabidopsis. In this method, we use protoplasts isolated from leaf tissues of Arabidopsis. Here, we provide a detailed protocol for using protoplasts to investigate the mechanism by which proteins are imported into chloroplasts.

Here we describe a protocol to express proteins into protoplasts by using PEG-mediated transformation method. The method provides easy expression of proteins of interest, and efficient investigation of protein localization and the import process for various experimental conditions in vivo.

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Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

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Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

Cell-free expression systems allow the tailored design of reaction environments to support the functional folding of even complex proteins such as membrane proteins. The experimental procedures for the co-translational insertion and folding of membrane proteins into preformed and defined membranes supplied as nanodiscs are demonstrated. The protocol is completely detergent-free and can generate milligrams of purified samples within one day. The resulting membrane protein/nanodisc samples can be used for a variety of functional studies and structural applications such as crystallization, nuclear magnetic resonance, or electron microscopy. The preparation of basic key components such as cell-free lysates, nanodiscs with designed membranes, critical stock solutions as well as the assembly of two-compartment cell-free expression reactions is described. Since folding requirements of membrane proteins can be highly diverse, a major focus of this protocol is the modulation of parameters and reaction steps important for sample quality such as critical basic reaction compounds, membrane composition of nanodiscs, redox and chaperone environment, or DNA template design. The whole process is demonstrated with the synthesis of proteorhodopsin and a G-protein coupled receptor.

Co-translational insertion into pre-formed nanodiscs makes it possible to study cell-free synthesized membrane proteins in defined lipid environments without contact with detergents. This protocol describes the preparation of essential system components and the critical parameters for improving expression efficiency and sample quality.