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 water ad libitum. The animals used in this study were 6-8 weeks old Sprague Dawley (SD) male rats, weighing about 250 g and specific pathogen-free. The animals were obtained from a commercial source (see Table of Materials).
1. Isolation of rat neutrophils
<ol>
	<li>Bone marrow harvesting
	<ol>
		<li>Place the rat in an appropriate container for anesthesia (see Table of Materials). Administer 3% isoflurane to the rat until the animal is unconscious. Check for the absence of a response to a toe pinch or tail pinch to ensure the depth of anesthesia.</li>
		<li>Once the rat is deeply anesthetized, perform cervical dislocation by placing the rat on its back and holding its tail with one hand while grasping the head with the other hand. Dislocate the neck quickly and firmly by applying a sudden and strong upward force on the tail while pulling the head downwards until the cervical spine is severed. Wait for confirmation of death, such as cessation of breathing and lack of heartbeat, before proceeding with tissue collection or disposal.</li>
		<li>Dip the rat in 75% ethanol and let it sit for a few minutes to sterilize the fur and skin. Lay the rat on its back and sever the lower limbs at the hip to protect the femur head. Use dissection scissors to cut through the ligament at the hock joint and fold the knee joint backward to expose the femur and tibia.</li>
		<li>Using forceps and scissors, carefully remove any muscles, tendons, and other tissues connected to the bones. Rinse the bones with Hank&#39;s Balanced Salt Solution (HBSS) to remove any remaining tissue debris and blood. Repeat two more times, for a total of three washes. Place the cleaned bones into a sterile container.</li>
		<li>Use a 5 or 10 mL syringe with a needle to poke the marrow through both ends of the bone to loosen the marrow matrix. Rinse the bone marrow with 10-15 mL Roswell Park Memorial Institute (RPMI) media with another syringe, until no visible bone marrow can be flushed out.</li>
		<li>Centrifuge the bone marrow cells at 300 x g for 10 min at 20 &#176;C. Add 5-10 mL red blood cell lysis buffer (see Table of Materials) to resuspend the cells and incubate at room temperature for 3 min. Add 4 times the volume of HBSS to the mixture. Centrifuge the cells at 600 x g for 5 min at room temperature, discard the supernatant with a pipette, and use the resulting pellet for further use.</li>
	</ol>
	</li>
	<li>Isolation of neutrophils from bone marrow
	<ol>
		<li>For the two-step method, perform the following additional steps:
		<ol>
			<li>Isolate bone marrow cells using the commercialized rat neutrophil isolation kit following the manufacturer&#39;s instructions (see Table of Materials).</li>
			<li>Prepare a density gradient by layering 2 mL of 55% percoll reagent (see Table of Materials), 2 mL of 65% of the reagent, 2 mL of 70% reagent, and 2 mL of 80% reagent in a new 15 mL centrifuge tube. Centrifuge the tube at 800 x g for 40 min at 20 &#176;C, with the acceleration set to 5 and the deceleration set to 0 or 1.</li>
			<li>Collect the 70% gradient layer, including the cell layers at the boundary between 65% and 70% gradient reagent, using a sterile pipette. Transfer the cell suspension to a new 15 mL centrifuge tube. Add 15 mL HBSS into the tube and gently invert the tube several times to wash the cells. Centrifuge the tube at 300 x g for 10 min at room temperature to pellet the cells.</li>
		</ol>
		</li>
		<li>For the one-step method, perform the following additional steps:
		<ol>
			<li>Subject bone marrow cells to the gradients without using the rat neutrophil isolation kit.</li>
			<li>Collect the 70% gradient layer, including the cell layers at the boundary between 65% and 70% gradient, using a sterile pipette. Transfer the cell suspension to a new 50 mL centrifuge tube and wash the cells with HBSS. Centrifuge the tube at 400 x g for 5 min at room temperature to pellet the cells.</li>
			<li>Count the isolated neutrophils using a hemocytometer and assess viability using trypan blue staining. 
			&#8203;NOTE: The protocol can be modified depending on the number of rats and the desired number of isolated neutrophils. An acceptable quantity of bones is obtained from three rats when this method is used for the first time in a new experimental setting. All procedures should be performed under sterile conditions. The isolation of neutrophils from bone marrow should be performed as quickly as possible to minimize cell damage and loss of viability.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Acquisition of rat NETs
<ol>
	<li>Resuspend 0.5 x 108-1 x 108 isolated neutrophils in 4 mL RPMI media (supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin) in a sterile 10 cm Petri dish.</li>
	<li>Add 500 nM PMA (see Table of Materials) to the neutrophil suspension to induce NETosis. Incubate for 3 h at 37 &#176;C and 5% CO2.</li>
	<li>For the negative control, add DNase I (10 U/mL) into the neutrophil suspension to degrade the secreted NETs.</li>
	<li>To harvest the NETs, remove the media and gently wash the NETs attached to the Petri dish with HBSS. Then, use intense flushing with 4 mL of fresh media for each plate to detach the NETs from the plate.</li>
	<li>Collect the washing medium and pipette frequently for complete resuspension of NETs.</li>
	<li>Centrifuge the suspension at 300 &#215; g for 10 min at 20 &#176;C to remove any floating cells.</li>
	<li>Transfer the suspension containing the NETs to a sterile tube and store at &#8722;20 &#176;C for further use within 2 weeks. 
	&#8203;NOTE: NETs are sensitive to degradation, so it is recommended to use freshly harvested material for best results.</li>
</ol>
3. Verification of the presence of NETs
<ol>
	<li>Fix the cells (from step 2.4) on coverslips with 4% paraformaldehyde for 15 min and NETs (from step 2.7) for 30 min to 1 h.</li>
	<li>Invert the coverslip onto an PBS/PBST droplet on a paraffin film-covered test tube stand and repeat the washing process thrice for 5 min each.</li>
	<li>Permeabilize the cells with 0.5% Triton-X-100 for 10 min at room temperature and wash three times in PBS/PBST for 1 min each. Seal the cells with the 10% normal donkey serum (see Table of Materials) at room temperature&#160;for 1 hour.</li>
	<li>Incubate the cells with the anti-rat neutrophil elastase antibody and anti-rat myeloperoxidase antibody (see Table of Materials) overnight at 4 &#176;C. Then, wash the coverslip in PBS/PBST three times for 5 min each.</li>
	<li>Incubate the cells with the secondary antibody A488-conjugated donkey anti-rabbit IgG (H + L), A594-conjugated donkey anti-mouse IgG (H + L), and A594-conjugated goat anti-Mouse IgG1 (see Table of Materials) at room temperature for 1 h in the dark. Then, wash the coverslip in PBS/PBST three times for 5 min each.</li>
	<li>Stain cell nuclei and NET skeletons with 10 mg/mL DAPI. Then, wash the coverslip in PBS/PBST three times for 5 min each.</li>
	<li>Place a drop of mounting medium on a glass slide and invert the coverslip with cells onto it. Let it dry for 1 h for microscopic analysis with immersion lenses; otherwise, it&#39;s ready for inspection. 
	&#8203;NOTE: Great care must be taken when manipulating NETs, even after fixation, as they are extremely fragile and can easily be lost during preparation.</li>
</ol>
4. Quantification of NETs
<ol>
	<li>Prepare Tris-EDTA (TE) buffer and PicoGreen (dsDNA assay reagent) working solution according to the manufacturer&#39;s instructions (see Table of Materials).</li>
	<li>Prepare standards by diluting stock DNA to concentrations of 1 ng/mL, 10 ng/mL, 100 ng/mL, and 1 &#181;g/mL. 
	NOTE: To quantify the concentration of NETs, the dsDNA assay kit (see Table of Materials) was employed, adhering to the manufacturer&#39;s guidelines. Tris-EDTA (TE) buffer and dsDNA assay reagents were readied using a 19-fold volume of double-distilled water or a 199-fold volume of TE buffer, respectively. Subsequently, standard solutions (1 ng/mL, 10 ng/mL, 100 ng/mL, and 1 &#181;g/mL) were prepared, and each 50 &#181;L of the sample was combined with 450 &#181;L of TE buffer.</li>
	<li>Add 50 &#181;L of each sample to 450 &#181;L TE buffer.</li>
	<li>Add 100 &#181;L of standards or samples along with an equal volume of assay reagent working solution to each well in a 96-well plate, including standards and samples.</li>
	<li>Incubate the plate at room temperature for 2-5 min, avoiding direct light.</li>
	<li>Read the samples using a fluorescence microplate reader with an emission spectrum of around 530 nm and an absorbance spectrum of around 480 nm.</li>
	<li>Calculate the concentration of NETs in each sample using the standard curve generated by the standards.</li>
</ol>
5. Analysis of NETs secretion by cell cytometry
<ol>
	<li>Add nucleus stain (see Table of Materials) to the cells incubated in the 96-well plate to reach a final concentration of 10 ng/mL and incubate for 30 min.</li>
	<li>Add cell-free DNA (cfDNA, see Table of Materials) stain to the cells to reach a final concentration of 300 nM and incubate for 10 min.</li>
	<li>Mix the fluorescent dyes by gentle pipetting. Do not discard the supernatant or wash the pellet.</li>
	<li>Load the sample plate into the cytometer instrument.</li>
	<li>Set the parameters for focus and exposure time for the different channels: blue (ex: 377/50 nm, em: 470/22 nm), usually with the exposure of 30,000 ms, green (ex: 483/32 nm, eM: 536/40 nm) usually with the exposure of 5,000 ms.</li>
	<li>Capture highly uniform images of the entire plate at different channels.</li>
	<li>Analyze the nucleus stain counts for different groups. If they are similar, NETs secretion can be determined by the cfDNA stain count.</li>
</ol>

Efficient Rat Bone Marrow Neutrophil Extracellular Trap Isolation

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	<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 authors have no conflicts of interest to declare.

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><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&amp;nbsp;</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&amp;rsquo;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>&amp;nbsp;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.

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

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

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.

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 ...

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Research

JoVE Journal

Biology

1.8K Views.  Sichuan University. We hebben een methode ontwikkeld om neutrofielen van ratten te isoleren en vervolgens NET's te extraheren. Deze methode biedt de mogelijkheid om de toegankelijkheid te optimaliseren voor het uitvoeren van NET-gerelateerde experimenten in zowel ratten als van ratten afgeleide cellijnen. In onze studie voerden we een vergelijking uit tussen neutrofielen die werden geïsoleerd uit het perifere bloed en de beenmergdraden.Onze bevindingen benadrukken de superioriteit van van beenmerg afgele...

Video: Auteur in de schijnwerpers: geoptimaliseerde methode voor het isoleren van rattenneutrofielen en NET's uit beenmerg

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

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

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 experiments.

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Immunology and Infection

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.
In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45&#8722; mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

Medicine

Scalable ATAC-seq Protocol for Chromatin Accessibility in Rodent Neutrophils

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

Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) is a powerful, high-throughput technique for assessing chromatin accessibility and understanding epigenomic regulation. Neutrophils, as a crucial leukocyte type in immune responses, undergo substantial chromatin architectural changes during differentiation and activation, which significantly impact the gene expression necessary for their functions. ATAC-seq has been instrumental in uncovering key transcription factors in neutrophil maturation, revealing pathogen-specific epigenomic signatures, and identifying therapeutic targets for autoimmune diseases. However, neutrophils' sensitivity to the external milieu complicates high-quality ATAC-seq data production. Here, we propose a scalable protocol for preparing ATAC-seq libraries from rodent bone marrow-derived neutrophils, featuring improved immunomagnetic separation to ensure optimal cell viability and high-quality libraries. The vital elements impacting the library quality and optimization principles for methodological extension are discussed in detail. This protocol will support the researchers who are willing to study the chromatin architecture and epigenomic reprogramming of neutrophils, advancing studies in basic and clinical immunology.

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 neutrophils.

Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) is a powerful, high-throughput technique for assessing chromatin accessibility and ...

Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method

With a decrease of bone mineral density, bones are more likely to fracture, thus negatively affecting a patient's quality of life. The growth and development of bones are mainly regulated by osteoblasts and osteoclasts. It has been widely accepted that osteoclasts are derived from bone marrow monocyte-macrophage cells (BMMs). BMMs and other hematopoietic stem cells are located in the bone marrow cavity. Therefore, isolating single stable BMMs from different and heterogeneous cell populations is a huge challenge. Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method. Adherent cells cultured for 24-48 h in primary culture were collected. Flow cytometric analysis showed that approximately 37.94% of the cells were CD11b/c+ (monocyte-macrophage surface antigen). Tartrate resistant acid phosphatase (TRAP) staining and western blot analysis demonstrated that BMMs could differentiate into osteoclasts in vitro. The above findings suggested that the secondary adherence cells could be considered as a suitable cellular model for osteoclast differentiation research.

Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method.

With a decrease of bone mineral density, bones are more likely to fracture, thus negatively affecting a patient's quality of life. The growth and ...

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

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

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 methods.

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Niche-Based Isolation of Rat Jaw Bone Marrow Mesenchymal Stem Cells

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

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a &#39;niche-based approach on stemness&#39; for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a &#39;fibroblast-like&#39; spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

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 rats.

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from ...

Unique Approach for Isolating Rat Bone Marrow Neutrophils with Comparable Capacity

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