Name: Oppkjøp og verifisering av Rat Neutrophil ekstracellulære feller (NET)
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)

Effektiv rottebenmarg nøytrofil ekstracellulær felleisolasjon

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.

Forfatter Spotlight: Optimalisert metode for å isolere rottenøytrofiler og NET fra benmarg

Unik tilnærming for isolering av rottebenmarg nøytrofiler med sammenlignbar kapasitet

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

Oppkjøp og verifisering av Rat Neutrophil ekstracellulære feller (NET)

For å begynne, resuspendere de isolerte rotte nøytrofilene i fire milliliter supplert RPMI-media i en steril 10-centimeter petriskål. Deretter legger du til fire mikroliter PMA til den nøytrofile suspensjonen for å utløse dannelsen av NET. Inkuber blandingen ved 37 grader Celsius i tre timer under 5% karbondioksid.For den negative kontrollen, tilsett fire til fem mikroliter DNASE1 til den nøytrofile suspensjonen for å nedbryte de utskilte NET. Deretter legger du til fire milliliter HBSS for å vaske NET-ene. Skyll platen med fire milliliter ferske kulturmedier for hver plate for å løsne NET-ene.Samle nå vaskemediet i et nytt rør og pipette ofte for å sikre fullstendig oppheng av NET-ene. Sentrifuger suspensjonen ved 300 g i 10 minutter ved 20 grader Celsius for å fjerne eventuelle flytende celler. Deretter samler du supernatantmediet med NET i et nytt rør.Sentrifuge suspensjonen av NET og flytende celler i en 24-brønns plate med 1,4 centimeter glassdeksel slips plassert i den for å avgjøre eventuelle flytende celler. Fest deretter cellene på dekselsedler med 4% PFA og fortsett med å bekrefte tilstedeværelsen av NET-ene. Legg deretter en dråpe HBSS på et parafinfilmdekket reagensrørstativ.Snu dekselet inn i HBSS-dråpene for å vaske det. Etter vask, inkuber cellene i 250 mikroliter 0,5x Triton X-100 i 10 minutter ved romtemperatur for å permeabilisere dem. Deretter vasker du cellene tre ganger i HBSS i ett minutt.Forsegle cellene i 10% normalt eselserum ved romtemperatur i en time. Til slutt, inkuber cellene med 70 til 90 mikroliter anti-rotte antistoffer over natten ved fire grader Celsius. Vask dekselet i HBSS tre ganger i fem minutter hver.Inkuber nå dekselet i sekundære antistoffer ved romtemperatur i en time i mørket før du vasker en HBSS. Flekk cellekjernene og NETs skjeletter i 70 til 90 mikroliter DAPI. Utfør HBSS vask tre ganger i fem minutter hver.

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 ...