Pioch Jonathan, Blomgran Robert
Division of Inflammation and Infection, Department of Biomedical and Clinical Sciences, Linköping University, Sweden.
Division of Inflammation and Infection, Department of Biomedical and Clinical Sciences, Linköping University, Sweden.
J Immunol Methods. 2022 Aug;507:113308. doi: 10.1016/j.jim.2022.113308. Epub 2022 Jun 26.
Reactive oxygen species (ROS) and the ability of immune cells to mount an oxidative burst represent an important defense during microbial invasion, but is also recognized for playing a significant role in the progression of inflammatory disorders and disease. Although neutrophils produce the strongest ROS-response, other leukocytes and their cell subsets could play a significant role. Isolation of specific cells for determining their ROS-response can affect their functionality and is laborious or hard to replicate in different settings. We have therefore established a whole blood assay, that only requires 100 μL heparinized blood and utilizes the dihydrorhodamine (DHR) 123 ROS-probe combined with cell surface antibody staining for the specific detection of ROS in several subsets of cells simultaneously using flow cytometry. Although the flow markers chosen are interchangeable with other direct conjugated and cell specific antibodies depending on the research question, we focused on neutrophils (SSCCD16HLA-DR), eosinophils (SSCCD16HLA-DRCD193CD125) and monocyte subsets (SSCHLA-DRCD14CD16). As a RBC-lysis reagent we compared BD FACS Lysis Solution to the in-house prepared ammonium-chloride‑potassium based ACK Lysis Buffer, that does not fix or permeabilize the immune cells. We find that ACK-lysis of stimulated and stained samples results in superior surface staining, decreased loss of cell subsets, and enhanced resolution of the DHR-signal. Compared to the other cells analyzed in healthy blood donors, neutrophils responded with the highest ROS-response to all tested stimuli (fMLP (low stimuli), E. coli, and PMA (high stimuli)), where eosinophils and the three monocyte subsets also showed an extensive ROS-response when stimulated with E. coli or PMA. Our assay provides the possibility for researchers to examine the ROS-response of specific cell subsets in specific patient groups ex vivo and could also allow the analysis of pharmacological intervention studies targeting ROS, which ultimately can advance the field of immunological research.
活性氧（ROS）以及免疫细胞产生氧化爆发的能力是微生物入侵过程中的重要防御机制，但在炎症性疾病和病症的进展中也起着重要作用。尽管中性粒细胞产生最强的ROS反应，但其他白细胞及其细胞亚群也可能发挥重要作用。分离特定细胞以确定其ROS反应会影响其功能，并且费力或难以在不同环境中重复。因此，我们建立了一种全血检测方法，该方法仅需100μL肝素化血液，并利用二氢罗丹明（DHR）123 ROS探针结合细胞表面抗体染色，通过流式细胞术同时特异性检测多个细胞亚群中的ROS。尽管根据研究问题选择的流式标记可与其他直接偶联的细胞特异性抗体互换，但我们重点关注中性粒细胞（SSCCD16HLA-DR）、嗜酸性粒细胞（SSCCD16HLA-DRCD193CD125）和单核细胞亚群（SSCHLA-DRCD14CD16）。作为红细胞裂解试剂，我们将BD FACS裂解液与内部制备的基于氯化铵-钾的ACK裂解缓冲液进行了比较，后者不会固定或通透免疫细胞。我们发现，对刺激和染色后的样品进行ACK裂解可产生更好的表面染色效果，减少细胞亚群的损失，并增强DHR信号的分辨率。与健康献血者中分析的其他细胞相比，中性粒细胞对所有测试刺激（fMLP（低刺激）、大肠杆菌和佛波酯（PMA，高刺激））的ROS反应最强，其中嗜酸性粒细胞和三个单核细胞亚群在用大肠杆菌或PMA刺激时也表现出广泛的ROS反应。我们的检测方法为研究人员提供了在体外检查特定患者群体中特定细胞亚群的ROS反应的可能性，也可用于分析针对ROS的药理干预研究，这最终可以推动免疫研究领域的发展。
