Name: Verwerving en verificatie van extracellulaire vallen (NET's) voor neutrofielen bij ratten
Uploaded: 2024-04-26T12:30:00
Description: Watch this Scientific Journal Video about Acquisition and Verification of Rat Neutrophil Extracellular Traps NETs at JoVE.com

acquisition and verification of rat neutrophil extracellular traps (nets)

Efficiënte isolatie van de Neutrofiele Extracellulaire Valstrik van het Rat Beenmerg

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.

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

Unieke aanpak voor het isoleren van beenmergneutrofielen bij ratten met vergelijkbare capaciteit

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 Acquisition and Verification of Rat Neutrophil Extracellular Traps NETs at JoVE.com

Verwerving en verificatie van extracellulaire vallen (NET's) voor neutrofielen bij ratten

Resuspendeer om te beginnen de geïsoleerde neutrofielen van ratten in vier milliliter aangevuld RPMI-medium in een steriele petrischaal van 10 centimeter. Voeg vervolgens vier microliter PMA toe aan de neutrofielensuspensie om de vorming van NET's op gang te brengen. Incubeer het mengsel bij 37 graden Celsius gedurende drie uur onder 5% koolstofdioxide.Voeg voor de negatieve controle vier tot vijf microliter DNASE1 toe aan de neutrofielensuspensie om de uitgescheiden NET's af te breken. Voeg vervolgens vier milliliter HBSS toe om de NET's te wassen. Spoel de plaat met vier milliliter verse kweekmedia voor elke plaat om de NET's los te maken.Vang nu het wasmedium op in een nieuwe tube en pipet regelmatig om ervoor te zorgen dat de NET's volledig opnieuw worden gesust. Centrifugeer de suspensie op 300 g gedurende 10 minuten bij 20 graden Celsius om eventuele zwevende cellen te verwijderen. Verzamel vervolgens het supernatans medium met NET's in een nieuwe buis.Centrifugeer de suspensie van NET's en drijvende cellen in een plaat met 24 putjes en glazen afdekglaasjes van 1,4 centimeter erin geplaatst om eventuele drijvende cellen te bezinken. Bevestig vervolgens de cellen op dekglaasjes met 4%PFA en ga verder met het verifiëren van de aanwezigheid van de NET's. Plaats vervolgens een druppel HBSS op een met paraffinefilm bedekte reageerbuisstandaard.Keer de afdekglaasje om in de HBSS-druppels om het te wassen. Na het wassen incubeer je de cellen in 250 microliter 0,5x Triton X-100 gedurende 10 minuten bij kamertemperatuur om ze te permeabiliseren. Was de cellen vervolgens drie keer in HBSS gedurende één minuut.Verzegel de cellen gedurende een uur in 10%normaal ezelserum bij kamertemperatuur. Tot slot, incubeer de cellen met 70 tot 90 microliter antilichamen tegen ratten 's nachts bij vier graden Celsius. Was het afdekglaasje in HBSS driemaal gedurende vijf minuten.Incubeer nu het afdekglaasje in de secundaire antilichamen bij kamertemperatuur gedurende een uur in het donker voordat u een HBSS wast. Kleurt de celkernen en NET-skeletten in 70 tot 90 microliter DAPI. Voer HBSS-wasbeurt drie keer uit gedurende elk vijf minuten.

Research

JoVE Journal

Biology

Acquisition and Verification of Rat 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 ...