Name: Método de uma e duas etapas de isolamento de neutrófilos em ratos para coleta de armadilhas extracelulares de neutrófilos (NETs)
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)

Isolamento eficiente da armadilha extracelular de neutrófilos da medula óssea de ratos

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 em destaque: Método otimizado para isolar neutrófilos de ratos e TNEs da medula óssea

Abordagem única para isolar neutrófilos de medula óssea de rato com capacidade comparável

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 uma e duas etapas de isolamento de neutrófilos em ratos para coleta de armadilhas extracelulares de neutrófilos (NETs)

Para iniciar a colheita da medula óssea, mergulhe um rato eutanasiado em etanol 75% para esterilizar o pelo e a pele. Deite o rato de costas e corte os membros inferiores no quadril para proteger a cabeça do fêmur. Use tesoura de dissecção para cortar o ligamento na articulação do hock.Em seguida, dobre a articulação do joelho para trás para expor o fêmur e a tíbia. Com um par de pinças e tesouras, remova cuidadosamente quaisquer músculos, tendões e outros tecidos conectados aos ossos. Cutuque a medula através de ambas as extremidades ósseas com uma seringa agulhada de 5 milímetros para soltar a matriz da medula.Agora use uma seringa fresca para limpar a medula óssea com 10-15 mililitros de mídia RPMI. Centrifugar as células da medula óssea a 300G por 10 minutos a 20 graus Celsius. Descarte o sobrenadante e ressuspenda as células em 5-10 mililitros de tampão de lise de hemácias.Após incubar a suspensão, adicionar quatro vezes o volume de HBSS à mistura. Em seguida, centrifugar as células a 600G por cinco minutos à temperatura ambiente. Use o pellet resultante para uso posterior.Isole os neutrófilos primeiro com kit de isolamento de neutrófilos de medula óssea de ratos. Camada 2 mililitros de 80-55% por col reagente em um tubo centrífugo de 15 mililitros, para criar um gradiente de densidade. Em seguida, pipetar os neutrófilos isolados, ou a solução de ressuspensão de células isoladas com kit de isolamento de neutrófilos de medula óssea de rato, para dentro do tubo.Centrifugar o tubo a 800G durante 40 minutos a 20 graus Celsius. Com uma pipeta estéril, colete a camada de gradiente de 70%, incluindo as camadas de células no limite de 65-70%. Transfira a suspensão da célula para um novo tubo centrífugo de 15 mililitros.Adicione 15 mililitros de HBSS no tubo e inverta suavemente para misturar seu conteúdo. Finalmente, centrifugar o tubo a 300G por 10 minutos à temperatura ambiente para pellet das células. Para o processo de uma etapa, após pipetar a suspensão da medula óssea diretamente no tubo de gradiente de densidade, centrifugar como demonstrado anteriormente.Em seguida, pipete a camada de gradiente de 70%, incluindo as camadas no limite de gradiente de 75-70%. Transfira esta suspensão celular para um novo tubo centrífugo de 50 mililitros com 10 mililitros de HBSS. Em seguida, centrifugar o tubo a 400G por cinco minutos à temperatura ambiente para peletizar as células.Contar os neutrófilos isolados usando um hemocitômetro e avaliar a viabilidade celular através da coloração com azul de tripano. O método de duas etapas teve um nível de pureza aumentado de 90% em relação aos 50% alcançados pelo método de uma etapa.

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 ...