Name: Método de uno y dos pasos de aislamiento de neutrófilos en ratas para la recolección de trampas extracelulares de neutrófilos (NET)
Uploaded: 2024-04-26T12:30:00
Description: Watch this Scientific Journal Video about One and Two-Step Method of Rat Neutrophil Isolation for Harvesting Neutrophil Extracellular Traps NETs at JoVE.com

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

Aislamiento eficiente de trampas extracelulares de neutrófilos en médula ósea de rata

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.

Autor destacado: Método optimizado para aislar neutrófilos y NET de rata de la médula ósea

Enfoque único para aislar neutrófilos de médula ósea de rata con capacidad comparable

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.

Watch this Scientific Journal Video about One and Two-Step Method of Rat Neutrophil Isolation for Harvesting Neutrophil Extracellular Traps NETs at JoVE.com

Método de uno y dos pasos de aislamiento de neutrófilos en ratas para la recolección de trampas extracelulares de neutrófilos (NET)

Para comenzar la recolección de médula ósea, remoje una rata sacrificada en etanol al 75% para esterilizar el pelaje y la piel. Coloca a la rata boca arriba y corta las extremidades inferiores a la altura de la cadera para proteger la cabeza del fémur. Use tijeras de disección para cortar el ligamento en la articulación del corvejón.A continuación, dobla la articulación de la rodilla hacia atrás para exponer el fémur y la tibia. Con un par de pinzas y tijeras, retire con cuidado los músculos, tendones y otros tejidos conectados a los huesos. Introduzca la médula a través de ambos extremos óseos con una jeringa agujereada de 5 milímetros para aflojar la matriz de la médula.Ahora use una jeringa nueva para enjuagar la médula ósea con 10-15 mililitros de medio RPMI. Centrifugar las células de la médula ósea a 300 g durante 10 minutos a 20 grados centígrados. Deseche el sobrenadante y vuelva a suspender las células en 5-10 mililitros de tampón de lisis de glóbulos rojos.Después de incubar la suspensión, agregue cuatro veces el volumen de HBSS a la mezcla. A continuación, centrifugar las células a 600 g durante cinco minutos a temperatura ambiente. Utilice el gránulo resultante para su uso posterior.Aísle primero los neutrófilos con el kit de aislamiento de neutrófilos de médula ósea de rata. Coloque una capa de 2 mililitros de 80-55% por reactivo en un tubo de centrífuga de 15 mililitros, para crear un gradiente de densidad. A continuación, pipetee los neutrófilos aislados, o la solución de resuspensión de las células aisladas con el kit de aislamiento de neutrófilos de médula ósea de rata, en el tubo.Centrifugar el tubo a 800 G durante 40 minutos a 20 grados centígrados. Con una pipeta estéril, recoja la capa de gradiente del 70 %, incluidas las capas celulares en el límite del 65-70 %. Transfiera la suspensión de la celda a un nuevo tubo de centrífuga de 15 mililitros.Agregue 15 mililitros de HBSS en el tubo e invierta suavemente para mezclar su contenido. Finalmente, centrifugar el tubo a 300 G durante 10 minutos a temperatura ambiente para granular las células. Para el proceso de un solo paso, después de pipetear la suspensión de médula ósea directamente en el tubo de gradiente de densidad, centrifugar como se demostró anteriormente.A continuación, pipetee la capa 70%gradient, incluidas las capas en el límite 75-70%gradiente. Transfiera esta suspensión de celda a un nuevo tubo de centrífuga de 50 mililitros con 10 mililitros de HBSS. A continuación, centrifugar el tubo a 400 g durante cinco minutos a temperatura ambiente para granular las células.Cuente los neutrófilos aislados con un hemocitómetro y evalúe la viabilidad celular mediante tinción con azul de tripano. El método de dos pasos tuvo un nivel de pureza mejorado del 90% en relación con el 50% logrado por el método de un solo paso.

Research

JoVE Journal

Biology

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

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 ...