Funding: This work was supported by the National Natural Science Foundation of China (Nos. 82004154, 81900311, 82100336 and 81970345).

The study was performed under a project license (No. 20211404A) granted by the Animal Ethics Committee of West China Hospital, Sichuan University, in compliance with the guidelines of the Animal Ethics Committee of West China Hospital, Sichuan University for the care and use of animals. In accordance with ethical guidelines, the rats used in this study were maintained in a controlled environment with a 12 h light/dark cycle, temperature at 22-24 &#176;C and humidity of 50%-60%. The rats were given access to food and wate...

Efficient Rat Bone Marrow Neutrophil Extracellular Trap Isolation

<ol>
	<li>Papayannopoulos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+in+immunity+and+disease.">Neutrophil extracellular traps in immunity and disease.</a> Nature Reviews Immunology. 18 (2), 134-147 (2018).</li><li>Takei, H., Araki, A., Watanabe, H., Ichinose, A., Sendo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+killing+of+human+neutrophils+by+the+potent+activator+phorbol+12-myristate+13-acetate+(PMA)+accompanied+by+changes+different+from+typical+apoptosis+or+necrosis.">Rapid killing of human neutrophils by the potent activator phorbol 12-myristate 13-acetate (PMA) accompanied by changes different from typical apoptosis or necrosis.</a> Journal of Leukocyte Biology. 59 (2), 229-240 (1996).</li><li>Brinkmann, V., 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=Neutrophil+extracellular+traps+kill+bacteria.">Neutrophil extracellular traps kill bacteria.</a> Science. 303 (5663), 1532-1535 (2004).</li><li>Li, T., 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=Neutrophil+extracellular+traps+induce+intestinal+damage+and+thrombotic+tendency+in+inflammatory+bowel+disease.">Neutrophil extracellular traps induce intestinal damage and thrombotic tendency in inflammatory bowel disease.</a> Journal of Crohn's and Colitis. 14 (2), 240-253 (2020).</li><li>Laridan, E., Martinod, K., De Meyer, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+in+arterial+and+venous+thrombosis.">Neutrophil extracellular traps in arterial and venous thrombosis.</a> Seminars in Thrombosis and Hemostasis. 45 (1), 86-93 (2019).</li><li>Dinallo, V., 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=Neutrophil+Extracellular+traps+sustain+inflammatory+signals+in+ulcerative+colitis.">Neutrophil Extracellular traps sustain inflammatory signals in ulcerative colitis.</a> Journal of Crohn's and Colitis. 13 (6), 772-784 (2019).</li><li>Dicker, A. 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=Neutrophil+extracellular+traps+are+associated+with+disease+severity+and+microbiota+diversity+in+patients+with+chronic+obstructive+pulmonary+disease.">Neutrophil extracellular traps are associated with disease severity and microbiota diversity in patients with chronic obstructive pulmonary disease.</a> Journal of Allergy and Clinical Immunology. 141 (1), 117-127 (2018).</li><li>Franck, G., 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=Roles+of+PAD4+and+netosis+in+experimental+atherosclerosis+and+arterial+injury:+Implications+for+superficial+erosion.">Roles of PAD4 and netosis in experimental atherosclerosis and arterial injury: Implications for superficial erosion.</a> Atherosclerosis. 275, e11 (2018).</li><li>Jorch, S. K., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+emerging+role+for+neutrophil+extracellular+traps+in+noninfectious+disease.">An emerging role for neutrophil extracellular traps in noninfectious disease.</a> Nature Medicine. 23 (3), 279-287 (2017).</li><li>Marin-Esteban, V., 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=Afa/Dr+diffusely+adhering+Escherichia+coli+strain+C1845+induces+neutrophil+extracellular+traps+that+kill+bacteria+and+damage+human+enterocyte-like+cells.">Afa/Dr diffusely adhering Escherichia coli strain C1845 induces neutrophil extracellular traps that kill bacteria and damage human enterocyte-like cells.</a> Infection and Immunity. 80 (5), 1891-1899 (2012).</li><li>Kang, L., 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=Neutrophil+extracellular+traps+released+by+neutrophils+impair+revascularization+and+vascular+remodeling+after+stroke.">Neutrophil extracellular traps released by neutrophils impair revascularization and vascular remodeling after stroke.</a> Nature Communications. 11 (1), 2488 (2020).</li><li>Sch&#252;ttler, D., 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=Animal+models+of+atrial+fibrillation.">Animal models of atrial fibrillation.</a> Circulation Research. 127 (1), 91-110 (2020).</li><li>Najmeh, S., Cools-Lartigue, J., Giannias, B., Spicer, J., Ferri, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+human+neutrophil+extracellular+traps+(NETs)+isolation+and+handling.">Simplified human neutrophil extracellular traps (NETs) isolation and handling.</a> Journal of Visualized Experiments. 98, e52687 (2015).</li><li>He, L., 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=Bone+marrow+is+the+preferred+source+for+isolation+of+rat+neutrophils+and+the+subsequent+acquisition+of+neutrophil+extracellular+traps.">Bone marrow is the preferred source for isolation of rat neutrophils and the subsequent acquisition of neutrophil extracellular traps.</a> Annals of Translational Medicine. 10 (15), 823-823 (2022).</li><li>Freeman, G. E., Dalton, C. A., Brooks, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Nycodenz+gradient+method+for+the+purification+of+neutrophils+from+the+peripheral+blood+of+rats.">A Nycodenz gradient method for the purification of neutrophils from the peripheral blood of rats.</a> Journal of Immunological Methods. 139 (2), 241-249 (1991).</li><li>Zindl, C. L., 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=IL-22-producing+neutrophils+contribute+to+antimicrobial+defense+and+restitution+of+colonic+epithelial+integrity+during+colitis.">IL-22-producing neutrophils contribute to antimicrobial defense and restitution of colonic epithelial integrity during colitis.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (31), 12768-12773 (2013).</li><li>Wong, K. L., 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=Gene+expression+profiling+reveals+the+defining+features+of+the+classical,+intermediate,+and+nonclassical+human+monocyte+subsets.">Gene expression profiling reveals the defining features of the classical, intermediate, and nonclassical human monocyte subsets.</a> Blood. 118 (5), e16-e31 (2011).</li><li>Nauseef, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+neutrophils+from+venous+blood.">Isolation of human neutrophils from venous blood.</a> Methods in Molecular Biology. 412, 15-20 (2007).</li><li>Lindena, J., Burkhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+chemiluminescence+properties+of+human,+canine+and+rat+polymorphonuclear+cells.">Separation and chemiluminescence properties of human, canine and rat polymorphonuclear cells.</a> Journal of Immunological Methods. 115 (1), 141-147 (1988).</li><li>Lauwers, M., 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=Optimization+of+the+Transwell+assay+for+the+analysis+of+neutrophil+chemotaxis+using+flow+cytometry+to+refine+the+clinical+investigation+of+immunodeficient+patients.">Optimization of the Transwell assay for the analysis of neutrophil chemotaxis using flow cytometry to refine the clinical investigation of immunodeficient patients.</a> Clinical Immunology. 238, 108994 (2022).</li><li>Evrard, M., 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=Developmental+analysis+of+bone+marrow+neutrophils+reveals+populations+specialized+in+expansion,+trafficking,+and+effector+functions.">Developmental analysis of bone marrow neutrophils reveals populations specialized in expansion, trafficking, and effector functions.</a> Immunity. 48 (2), 364-379 (2018).</li></ol>

The isolation of neutrophils constitutes a pivotal step in studying NETosis, where the selection of an appropriate isolation method is paramount for obtaining dependable results. An important factor to weigh is the occurrence of lymphocyte contamination during isolation. Addressing this challenge is particularly significant when isolating rat neutrophils from bone marrow. Despite the distinct density range of neutrophils (1.0814-1.0919, with a peak at 1.0919) compared to lymphocytes (1.0337-1.0765, with a peak at 1.0526)...

Neutrophils constitute a critical subset of leukocytes that play a pivotal role in the innate immune response. They are characterized by multilobed nuclei and granules containing various proteases and antimicrobial peptides1. Neutrophils primarily function through degranulation, phagocytosis, and the formation of NETs. The observation of NETs was first made by Takei et al. in 1996 during an experiment where neutrophils were stimulated with phorbol myristate acetate (PMA)2. Subsequently, the process of NET formation was coined &#34;NETosis&#34; by Brinkmann et al.3 in 2004. Their research further illuminated the crucial role of NETs in neutrophil-mediated antimicrobial responses. NETs are web-like structures composed of chromatin, histones, and antimicrobial proteins that are released from activated neutrophils in response to infectious and inflammatory stimuli. NETs can immobilize and kill invading pathogens by trapping them and exposing them to a high concentration of antimicrobial peptides and proteases1,3. Additionally, NETs contribute to the clearance of apoptotic cells and participate in inflammation resolution. Recent studies also indicate that an excessive formation of NETs or impaired NET degradation can lead to tissue damage, autoimmune disorders, thrombogenesis, and impaired revascularization4,5,6,7,8,9,10.
The pathogenic role of NETs in uncontrolled fibrosis following myocardial infarction and the formation of ventricular aneurysms has been demonstrated through the expansion of perivascular fibrosis4,11. The myocardial infarction model and the isolation of neutrophils from bone marrow in mice are both well-established. Polymorphonuclear (PMN) leukocytes, a type of white blood cell abundant in human blood, serve as an excellent source for isolating human neutrophils. This method eliminates the need to harvest bone marrow, thus enhancing safety and efficiency.
NETs also play a role in atrial fibrillation associated with cardiac remodeling. However, large animals such as dogs and pigs were utilized to model atrial fibrillation, as mice lack an atrium sizable enough to establish a re-entrant cycle or the AF model, unless specific ion channels or signaling pathways are knocked down or knocked out12. While it&#39;s possible to induce atrial fibrillation in rats and isolate neutrophils from rat peripheral blood as previously described, researchers encountered a limitation whereby only 2 x 105-5 x 105 neutrophils could be isolated from peripheral blood (10 mL per rat). Extracting sufficient NETs at each time point required approximately 10-25 rats (5 x 106 neutrophils in total), resulting in a time-consuming, expensive, and often low-yield process13. In this regard, Li He and colleagues present a bone marrow-oriented strategy to obtain adequate NETs from rats14. In their article, they provide a comprehensive description of isolating neutrophils from rat bone marrow and compare the NET secretion capabilities of rat peripheral and bone marrow neutrophils. The two methods outlined cater to distinct experimental goals, both resulting in sufficient quantities of rat bone marrow neutrophils while reducing the number of required rats. The two-step isolation method demonstrated superior neutrophil purification, while the one-step method proved time-efficient with acceptable purification levels. Furthermore, the researchers compared NETosis and NET formation between rat bone marrow neutrophils and their peripheral counterparts, finding equal potency with PMN. These findings significantly contribute to neutrophil-related studies of atrial fibrillation and underscore the importance of flexibly selecting different sources for neutrophil isolation in various experimental animals with differing neutrophil distributions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A488-conjugated donkey antirabbit IgG(H + L)</td>
			<td>Invitrogen, USA</td>
			<td>A32790</td>
			<td></td>
		</tr>
		<tr>
			<td>A594-conjugated donkey anti-mouse IgG(H + L)</td>
			<td>Invitrogen, USA</td>
			<td>A32744</td>
			<td></td>
		</tr>
		<tr>
			<td>A594-conjugated goat anti-Mouse IgG1&#160;</td>
			<td>Invitrogen, USA</td>
			<td>A21125</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rat myeloperoxidase</td>
			<td>Abcam, England</td>
			<td>ab134132</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rat neutrophil elastase</td>
			<td>Abcam, England</td>
			<td>ab21595</td>
			<td></td>
		</tr>
		<tr>
			<td>Celigo Image Cytometer</td>
			<td>Nexelom, USA</td>
			<td>200-BFFL-5C</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma, USA</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum (FBS)</td>
			<td>Gibco, USA</td>
			<td>10099141C</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco, USA</td>
			<td>C14175500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Thermofisher, USA</td>
			<td>33342</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD, China</td>
			<td>R510-22-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol</td>
			<td>Sigma, USA</td>
			<td>81381</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Solarbio, China</td>
			<td>SL050</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>biosharp, China</td>
			<td>BL539A</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Hyclone, USA</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE, USA</td>
			<td>P8370-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma, USA</td>
			<td>&#160;P1585</td>
			<td></td>
		</tr>
		<tr>
			<td>Picogreen dsDNA Assay Kit</td>
			<td>Invitrogen, USA</td>
			<td>P11496</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat neutrophil isolation kit</td>
			<td>Solarbio, China</td>
			<td>P9200</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysis buffer</td>
			<td>Solarbio, China</td>
			<td>R1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) media</td>
			<td>Hyclone, USA</td>
			<td>SH30809.01B</td>
			<td></td>
		</tr>
		<tr>
			<td>RWD Universal Animal Anesthesia Machine</td>
			<td>RWD, China</td>
			<td>R500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley (SD) rats</td>
			<td>Dashuo, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SytoxGreen</td>
			<td>Thermofisher, USA</td>
			<td>S7020</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-EDTA (TE) buffer</td>
			<td>Solarbio, China</td>
			<td>T1120</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Biofroxx, German</td>
			<td>1139ML100</td>
			<td></td>
		</tr>
	</tbody>
</table>

unique approach for isolating rat bone marrow neutrophils with comparable capacity

The primary aim of this research was to develop a reliable and efficient approach for isolating neutrophil extracellular traps (NETs) from rat bone marrow. This effort arose due to limitations associated with the traditional method of extracting NETs from peripheral blood, mainly due to the scarcity of available neutrophils for isolation. The study revealed two distinct methodologies for obtaining rat neutrophils from bone marrow: a streamlined one-step procedure that yielded satisfactory purification levels, and a more time-intensive two-step process that exhibited enhanced purification efficiency. Importantly, both techniques yielded a substantial quantity of viable neutrophils, ranging between 50 to 100 million per rat. This efficiency mirrored the results obtained from isolating neutrophils from both human and murine sources. Significantly, neutrophils derived from rat bone marrow exhibited comparable abilities to secrete NETs when compared with neutrophils obtained from peripheral blood. However, the bone marrow-based method consistently produced notably larger quantities of both neutrophils and NETs. This approach demonstrated the potential to obtain significantly greater amounts of these cellular components for further downstream applications. Notably, these isolated NETs and neutrophils hold promise for a range of applications, spanning the realms of inflammation, infection, and autoimmune diseases.

Author Spotlight: Optimized Method for Isolating Rat Neutrophils and NETs from Bone Marrow

Unique Approach for Isolating Rat Bone Marrow Neutrophils with Comparable Capacity

We have developed a method of isolating rat neutrophils and subsequently extracting NETs. This method offers to optimize the accessibility for conducting NETs-related experiments in both rats and rat-derived cell lines. In our study, we conducted a comparation between neutrophils that were isolated from the peripheral blood and bone marrow threads.Our findings highlight the superiority of bone marrow-derived neutrophils, showcasing a substantial numerical advantage in isolated neutrophil counts alongside their proficient capability to synchronize. Our bone marrow-based method for isolating rat neutrophils and NETs fills a research gap by optimizing isolation techniques for these cell types. Unlike previous methods that primarily used peripheral blood, our protocol targets bone marrow, increasing neutrophil in large yields, and offering a more accurate rat model for studying neutrophil and rat biology.

The protocol outlined herein delineates two distinct methods, each characterized by improved purification or streamlined steps. Both methods yielded approximately 0.5 x 108-1 x 108 neutrophils per rat. Flow cytometry analysis, employing the annexin V-FITC/PI apoptosis detection kit, exhibited cell viability above 90%, comparable to mouse and human counterparts (Figure 1). While lymphocyte contamination seemed inevitable during neutrophil isolation from bone marrow, the ...

Watch this Scientific Journal Video about Author Spotlight: Optimized Method for Isolating Rat Neutrophils and NETs from Bone Marrow at JoVE.com

This research outlines two techniques for isolating abundant neutrophil extracellular traps (NETs) from rat bone marrow. One method combines a commercial neutrophil isolation kit with density gradient centrifugation, while the other employs only density gradient centrifugation. Both approaches yield functional NETs surpassing those from peripheral blood neutrophils.

The primary aim of this research was to develop a reliable and efficient approach for isolating neutrophil extracellular traps (NETs) from rat bone ...

unique-approach-for-isolating-rat-bone-marrow-neutrophils-with

Research

JoVE Journal

Biology

1.2K ビュー.  Sichuan University. ラット好中球を単離し、NETを抽出する方法を開発しました。この方法は、ラットとラット由来の細胞株の両方でNETs関連の実験を実施するためのアクセシビリティを最適化することを提供します。本研究では、末梢血から単離した好中球と骨髄糸を比較しました。我々の知見は、骨髄由来の好中球の優位性を強調し、単離された好中球数において、その熟練した同期...

ビデオ: 著者スポットライト:骨髄からラット好中球およびNETを分離するための最適化された方法(英語)

This research outlines two techniques for isolating abundant neutrophil extracellular traps (NETs) from rat bone marrow. One method combines a commercial neutrophil isolation kit with density gradient centrifugation, while the other employs only density gradient centrifugation. Both approaches yield functional NETs surpassing those from peripheral blood...

https://cloudfront.jove.com/CDNSource/teasers/65506_b.jpg

neonatal mouse bone marrow isolation and preparation of bone marrow-derived macrophages

Author Spotlight: Developing a Reproducible Method for Isolating Bone Marrow-Derived Macrophages from Neonatal Mice to Advance Early Life Immune Responses

This protocol describes a non-enzymatic and straightforward method for isolating 7-9-day-old neonatal mouse bone marrow cells and generating differentiated macrophages using a supernatant of L929 cells as a source of granulocyte colony-stimulating factor (M-CSF). The bone marrow-derived macrophages were further analyzed for surface antigens F4/80, CD206, CD11b, and functional...

Phagocytic Assay Evaluation of Neonatal Bone Marrow Derived Macrophages 

https://cloudfront.jove.com/CDNSource/teasers/66613_b.jpg

non-enzymatic method for isolating neonatal mouse bone marrow cells

Non-Enzymatic Method for Isolating Neonatal Mouse Bone Marrow Cells

isolating mesangiogenic progenitor cells (mpcs) from human bone marrow

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

atac-seq library preparation of murine bone marrow-derived neutrophils

Author Spotlight: Optimizing ATAC-seq Library Construction for Investigating Neutrophil Chromatin Accessibility and Epigenetic Reprogramming in Rodent Models

This manuscript outlines a protocol for preparing an ATAC-seq library of neutrophils from murine bone marrow, aiming to guarantee optimal neutrophil viability and high library quality. It offers step-by-step instructions on BMC preparation, immunomagnetic sorting, and library construction, serving as a valuable guide, especially for newcomers to studying...

ATAC-Seq Library Preparation of Murine Bone Marrow-Derived Neutrophils

economical and efficient protocol for isolating and culturing bone marrow-derived dendritic cells from mice

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL...

isolation, purification, and differentiation of osteoclast precursors from rat bone marrow

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional...

isolation, purification and labeling of mouse bone marrow neutrophils for functional studies and adoptive transfer experiments

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking...

Scalable ATAC-seq Protocol for Chromatin Accessibility in Rodent Neutrophils

https://cloudfront.jove.com/CDNSource/teasers/67490_b.jpg

bone marrow-derived dendritic cells generation: a method to generate dendritic cells from mouse bone marrow

This video describes a protocol for the generation of dendritic cells--key antigen-presenting cells that activate T cells--from the bone marrow of an adult...

Bone Marrow-Derived Dendritic Cells Generation: A Method to Generate Dendritic Cells from Mouse Bone Marrow

an optimized method for isolating and expanding invariant natural killer t cells from mouse spleen

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

technique for isolation and culture of rat jaw bone marrow mesenchymal stem cells

Author Spotlight: Application of High-Purity Jawbone Marrow Mesenchymal Stem Cells in Tissue Engineering

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in...

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

identifying bone marrow microenvironmental populations in myelodysplastic syndrome and acute myeloid leukemia

Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

https://cloudfront.jove.com/CDNSource/teasers/66093_b.jpg

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

identification of peptides of small extracellular vesicles from bone marrow-derived macrophages

Author Spotlight: Peptidome Extraction from Small Extracellular Vesicles Isolated from Bone Marrow-Derived Macrophages 

This protocol describes a procedure to isolate small extracellular vesicles from macrophages by differential ultracentrifugation and extract the peptidome for identification by mass...

Isolating Small Extracellular Vesicles and Extracting Their Peptidome

one and two-step method of rat neutrophil isolation for harvesting neutrophil extracellular traps (nets)

One and Two-Step Method of Rat Neutrophil Isolation for Harvesting Neutrophil Extracellular Traps (NETs)

simplified intrafemoral injections using live mice allow for continuous bone marrow analysis

Author Spotlight: A Technique for Engraftment of Cells into the Mouse Femur for Bone Marrow Studies

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone...

Intrafemoral Injection for Bone Marrow Study in Live Mice

https://cloudfront.jove.com/CDNSource/teasers/65874_b.jpg

bone marrow aspiration: a method to obtain bone marrow to examine cell morphology

This video describes the technique of aspirating bone marrow from iliac spine and smear preparation for biopsy. In the example, we perform bone marrow aspiration from an acute myeloid leukemia (AML) patient and examine the cells to check for measurable residual disease (MRD), an important prognostic...