Researchers routinely use antibodies to assess the expression levels of proteins on the surface or intracellularly in a variety of different cell types. In this current study we highlight the importance of careful validation of antibodies for analysis of protein expression by flow cytometry and how failure to do so can significantly impact the interpretation of the data generated leading to false-positive results. There has been increasing awareness of the role the programmed death receptor 1 (PD-1) pathway plays in health and disease and a potential that programme death ligand 1 (PD-L1) may play a role in inflammatory disease. We aimed to investigate PD-L1 expression on human neutrophils isolated from healthy individuals and patients diagnosed with chronic obstructive pulmonary disease (COPD). We observed an increase in surface expression of PD-L1 by human neutrophils when incubated with AlexaFluor™700-conjugated anti-CD16. Through careful interrogation and antibody validation, we found a novel interaction between a commercially available anti-PD-L1 antibody and the AlexaFluor™700 fluorophore, resulting in this observed increase in PD-L1 signal. Surface expression of PD-L1 was not observed on neutrophils from healthy volunteers or patients with COPD when clone 29E.2A3 of anti-PD-L1 was not used with AlexaFluor™700-conjugated anti-CD16. This highlights the importance of robust antibody validation to ensure antibody compatibility in the context of multi-parametric flow cytometry panels. We also show that, without these validation experiments, novel neutrophil phenotypes could be falsely reported – an important consideration when there is increasing interest in neutrophil heterogeneity.

研究人员通常使用抗体来评估各种不同细胞类型中表面或细胞内蛋白质的表达水平。在当前的研究中，我们强调了仔细验证抗体以通过流式细胞仪分析蛋白质表达的重要性，以及这样做的失败会如何显着影响所生成数据的解释，从而导致假阳性结果。人们对程序性死亡受体1（PD-1）通路在健康和疾病中所起的作用的认识不断提高，并且程序性死亡配体1（PD-L1）在炎症性疾病中可能发挥作用。我们旨在研究从健康个体和诊断为慢性阻塞性肺疾病（COPD）的患者中分离出的人类嗜中性粒细胞中PD-L1的表达。当与AlexaFluor™700偶联的抗CD16孵育时，我们观察到人嗜中性粒细胞的PD-L1表面表达增加。通过仔细的询问和抗体验证，我们发现了市售抗PD-L1抗体与AlexaFluor™700荧光团之间的新型相互作用，从而导致观察到的PD-L1信号增加。当抗PD-L1的克隆29E.2A3未与缀有AlexaFluor™700的抗CD16一起使用时，健康志愿者或COPD患者的中性粒细胞未观察到PD-L1的表面表达。这突显了在多参数流式细胞仪面板中进行可靠抗体验证以确保抗体相容性的重要性。我们还表明，如果没有这些验证实验，