Macrophage extracellular traps (METs) represent a novel defense mechanism in the antimicrobial arsenal of macrophages. However, mechanisms of MET formation are still poorly understood and this is at least partially due to the lack of reliable and reproducible models. Thus, we aimed at establishing a protocol of MET induction by bone marrow–derived macrophages (BMDMs) obtained from cryopreserved and then thawed bone marrow (BM) mouse cells. We report that BMDMs obtained in this way were morphologically (F4/80⁺) and functionally (expression of inducible nitric oxide (NO) synthase and NO production) differentiated and responded to various stimuli of bacterial (lipopolysaccharide, LPS), fungal (zymosan) and chemical (PMA) origin. Importantly, BMDMs were successfully casting METs composed of extracellular DNA (extDNA) serving as their backbone to which proteins such as H2A.X histones and matrix metalloproteinase 9 (MMP-9) were attached. In rendered 3D structure of METs, extDNA and protein components were embedded in each other. Since studies had shown the involvement of oxygen species in MET release, we aimed at studying if reactive nitrogen species (RNS) such as NO are also involved in MET formation. By application of NOS inhibitor — L-NAME or nitric oxide donor (SNAP), we studied the involvement of endogenous and exogenous RNS in traps release. We demonstrated that L-NAME halted MET formation upon stimulation with LPS while SNAP alone induced it. The latter phenomenon was further enhanced in the presence of LPS. Taken together, our findings demonstrate that BMDMs obtained from cryopreserved BM cells are capable of forming METs in an RNS-dependent manner.

巨噬细胞胞外陷阱（MET）代表了巨噬细胞抗菌库中的一种新型防御机制。然而，人们对 MET 形成的机制仍然知之甚少，这至少部分是由于缺乏可靠和可重复的模型。因此，我们的目的是建立一种通过从冷冻保存然后解冻的骨髓 (BM) 小鼠细胞中获得的骨髓源性巨噬细胞 (BMDM) 诱导 MET 的方案。我们报告以这种方式获得的 BMDM 在形态上（F4/80 ⁺）和功能上（诱导型一氧化氮（NO）合酶的表达和 NO 的产生）是分化的，并对细菌（脂多糖，LPS）、真菌（酵母聚糖）的各种刺激有反应和化学（PMA）来源。重要的是，BMDM 成功铸造了由细胞外 DNA (extDNA) 组成的 MET，作为其主链，其上附着有 H2A.X 组蛋白和基质金属蛋白酶 9 (MMP-9) 等蛋白质。在 MET 的渲染 3D 结构中，extDNA 和蛋白质成分相互嵌入。由于研究表明氧物质参与 MET 释放，因此我们旨在研究活性氮物质 (RNS)（例如 NO）是否也参与 MET 形成。通过应用NOS抑制剂L-NAME或一氧化氮供体(SNAP)，我们研究了内源性和外源性RNS在陷阱释放中的参与。我们证明 L-NAME 在 LPS 刺激下阻止 MET 形成，而 SNAP 单独诱导它。后一种现象在 LPS 存在下进一步增强。总而言之，我们的研究结果表明，从冷冻保存的 BM 细胞中获得的 BMDM 能够以 RNS 依赖性方式形成 MET。