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 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.
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 "NETosis" 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'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.
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 °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
2. Acquisition of rat NETs
3. Verification of the presence of NETs
4. Quantification of NETs
5. Analysis of NETs secretion by cell cytometry
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 two-step method demonstrated an enhanced purity level of 90% as compared to the 50% achieved by the one-step method (Figure 2).
Comparable responses in NET secretion were discernible between peripheral and bone marrow neutrophils, irrespective of PMA stimulation (Figure 3). Furthermore, rat NETs exhibited limited cross-linking capabilities with one another (Figure 4). In an effort to delve deeper into the process of rat NETosis, PMA was employed as an inducer. NETs were detected via cfDNA staining, and comprehensive images were captured using a Cell Imaging Analyzer. Notably, rat neutrophils exhibited a higher propensity for spontaneous NETosis in contrast to mouse and human neutrophils, even without external stimulation. Incubation with PMA led to a 10% increase in cfDNA content after 4 h (Figure 5). When exposed to 500 nM PMA, the bone marrow of each rat yielded a final concentration of 8-12 µg/mL NET-DNA. It's noteworthy that intracellular contents were incompletely extruded in rat neutrophils during NETosis, resulting in the observation of numerous intracellular components. Additionally, rat NETs exhibited an inclination to form a gauze-like film due to enhanced adhesion tendencies, presenting a distinct cloud-like appearance.
Figure 1: Neutrophil viability assessment from bone marrow isolation. The optimal source for rat neutrophil isolation is bone marrow, which facilitates subsequent neutrophil extracellular trap acquisition. This figure is adapted from He et al.14. Please click here to view a larger version of this figure.
Figure 2: Neutrophil purification from peripheral blood and bone marrow via Wright-Giemsa staining. (A) Peripheral blood origin. (B) Bone marrow origin via the one-step method. (C) Bone marrow origin via the two-step method. Bone marrow extraction serves as the preferred approach for rat neutrophil isolation and the ensuing acquisition of neutrophil extracellular traps. Scale bar = 20 µm. Magnification = 400x. This figure is adapted from He et al.14. Please click here to view a larger version of this figure.
Figure 3: NET secretion upon incubation with PMA or PMA + DNase I. Bone marrow extraction represents the preferred route for rat neutrophil isolation and the subsequent collection of neutrophil extracellular traps. This figure is adapted from He et al.14. Please click here to view a larger version of this figure.
Figure 4: Immunofluorescent analysis. Immunofluorescent staining of the nucleus (blue, A), MPO (green, B), and merge image (C). NET: Neutrophil Extracellular Trap; MPO: Myeloperoxidase. Bone marrow extraction is the favored method for rat neutrophil isolation and the subsequent gathering of neutrophil extracellular traps. Scale bar = 50 µm. Magnification = 200x. This figure is adapted from He et al.14. Please click here to view a larger version of this figure.
Figure 5: Comprehensive analysis of cfDNA via cell imaging analyzer under various conditions. Fluorescent staining of cfDNA (green) and nucleus (blue). Bone marrow extraction is the primary approach for rat neutrophil isolation and the ensuing collection of neutrophil extracellular traps. Scale bar = 500 µm. This figure is adapted from He et al.14. Please click here to view a larger version of this figure.
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), contamination with lymphocytes remains inescapable. This can be attributed partly to the abundance of lymphocytes in the bone marrow and the augmented density of immature lymphocytes15. Techniques such as density gradient separation, such as using Percoll and Ficoll, can assist in curbing lymphocyte contamination, although achieving complete lymphocyte elimination may be challenging. A specialized Percoll solution, calibrated to a specific density gradient, can be employed to minimize contamination by capitalizing on the density variance between rat neutrophils and other bone marrow cells. Therefore, researchers should judiciously assess the potential impact of lymphocyte contamination on their experimental outcomes and take measures to mitigate its influence.
This study underscores the significance of tailoring isolation methods to match specific experimental requisites. In the method delineated previously14, the one-step method demonstrated being less demanding in terms of time and effort. As such, it was endorsed for NET acquisition due to the inconsequential impact of contaminating lymphocytes on NET secretion. These lymphocytes could be subsequently eliminated during the final centrifugation stage. Conversely, for endeavors entailing neutrophil isolation and subsequent evaluation of NETosis and NET secretion, the two-step method was recommended14. This approach allowed precise quantification of NETs produced by neutrophils while minimizing the influence of potential confounding factors stemming from other cell contaminations.
Modifications can be made to the isolation protocol to enhance both yield and purity. For instance, the utilization of an optimized density gradient reagent set can yield more neutrophils. Gentle pipetting and washing methods can likewise mitigate cell loss and bolster purity. Troubleshooting actions such as monitoring buffer pH and temperature, alongside adjusting centrifugation parameters, can effectively address challenges encountered during the isolation process. An associated constraint of bone marrow isolation lies in the likelihood of immature neutrophil and other bone marrow cell contamination, thereby impacting both purity and yield. Devising a streamlined approach to expel lymphocytes could enhance overall isolation efficiency. Another constraint pertains to the necessity of animal sacrifice for bone marrow isolation, which might be unsuitable for longitudinal investigations into rat neutrophils in vivo.
Neutrophil isolation for humans predominantly relies on peripheral blood. Conventional methods encompass Ficoll-Paque, Percoll, and immunomagnetic bead separation16,17,18. The Ficoll approach segregates leukocyte populations based on buoyancy and relies on a contrast agent to differentiate neutrophils. It offers simplicity and cost-effectiveness but compromises on purity and yield and presents challenges in eliminating red blood cells. On the other hand, Percoll exploits density gradients, yielding greater neutrophil purity and yield at the cost of specialized equipment and increased expenses and time19. Immunomagnetic bead separation is a newer, more specific technique utilizing antibody-conjugated magnetic beads that specifically bind to neutrophils with minimal contamination from other leukocytes. Although amenable to automation, it necessitates specialized equipment and incurs higher costs due to bead expenses. When selecting a neutrophil isolation method, researchers must weigh yield, purity, cost, and complexity. Currently, the most convenient method for human neutrophil isolation involves using PolymorphPrep20. This approach yields highly purified neutrophils in significant quantities within a short span. PolymorphPrep's principle hinges on cell separation according to density.
In mice, isolating neutrophils from peripheral blood is inadvisable due to low blood volumes and the challenge of securing sufficient quantity and purity for downstream experiments. Despite rats providing sufficient blood quantities (10 mL per rat), their peripheral blood characteristics differ from humans and mice. Rats inherently exhibit deficiencies in neutrophil extraction, with monocytes being the most abundant nucleated cells13,14. Hence, peripheral blood isolation only furnishes 2 x 105-5 x 105 neutrophils from a single rat. Bone marrow serves as a more viable neutrophil source, offering a readily available and abundant supply regardless of the animal's infection status. Alternatively, inducing an inflammatory environment in the abdominal cavity or thorax to enhance neutrophil infiltration for isolation is unreliable and intricate. As such, bone marrow extraction emerges as a practical, dependable route for rat neutrophil isolation21.
NETosis involves diverse cellular processes encompassing DNA decondensation, autophagy, and intracellular matrix expulsion1. In humans, NET extrusion is comprehensive, leaving behind remnants of the cell membrane. Intriguingly, rat neutrophil studies exhibit divergent patterns, revealing more intracellular content post-stimulation. Despite PMA stimulation, rat neutrophils exhibit incomplete NET extrusion, aligning with their spontaneous NETosis. Curiously, rat NETs demonstrate a proclivity for aggregated forms (aggNETs) rather than extensive network structure, potentially due to reduced cross-linking ability. This phenomenon could significantly impact neutrophil aggregation, influencing the broader immune response in rats. Future applications of bone marrow isolation could involve probing distinct signaling pathways and immune cells in NETosis. Additionally, this method can facilitate NETosis exploration in varied disease models, unraveling neutrophils' roles across different pathologies. Ultimately, novel techniques for isolating and characterizing NETs hold promise for unraveling the mechanisms driving NETosis and its functional implications across diverse animal models.
The authors have nothing to disclose.
Funding: This work was supported by the National Natural Science Foundation of China (Nos. 82004154, 81900311, 82100336 and 81970345).
A488-conjugated donkey antirabbit IgG(H + L) | Invitrogen, USA | A32790 | |
A594-conjugated donkey anti-mouse IgG(H + L) | Invitrogen, USA | A32744 | |
A594-conjugated goat anti-Mouse IgG1 | Invitrogen, USA | A21125 | |
Anti-rat myeloperoxidase | Abcam, England | ab134132 | |
Anti-rat neutrophil elastase | Abcam, England | ab21595 | |
Celigo Image Cytometer | Nexelom, USA | 200-BFFL-5C | |
DNase I | Sigma, USA | 10104159001 | |
fetal bovine serum (FBS) | Gibco, USA | 10099141C | |
Hank’s Balanced Salt Solution (HBSS) | Gibco, USA | C14175500BT | |
Hoechst | Thermofisher, USA | 33342 | |
Isoflurane | RWD, China | R510-22-10 | |
Mowiol | Sigma, USA | 81381 | |
Normal Donkey Serum | Solarbio, China | SL050 | |
Paraformaldehyde | biosharp, China | BL539A | |
Penicillin-streptomycin | Hyclone, USA | SV30010 | |
Percoll | GE, USA | P8370-1L | |
Phorbol 12-myristate 13-acetate (PMA) | Sigma, USA | P1585 | |
Picogreen dsDNA Assay Kit | Invitrogen, USA | P11496 | |
Rat neutrophil isolation kit | Solarbio, China | P9200 | |
Red blood cell lysis buffer | Solarbio, China | R1010 | |
Roswell Park Memorial Institute (RPMI) media | Hyclone, USA | SH30809.01B | |
RWD Universal Animal Anesthesia Machine | RWD, China | R500 | |
Sprague Dawley (SD) rats | Dashuo, China | ||
SytoxGreen | Thermofisher, USA | S7020 | |
Tris-EDTA (TE) buffer | Solarbio, China | T1120 | |
Triton-X-100 | Biofroxx, German | 1139ML100 |