优化CD115稳定性因素以精确检测小鼠单核细胞及其亚群

Stabilized CD115 by Optimizing Sample Processing and Accurate Analysis of Murine Monocyte and its Subsets

单核细胞是机体防御系统的重要组成部分,单核细胞数量发生改变一定程度上可反映机体炎症或其他疾病状态。应用流式细胞术对单核细胞表面抗原进行检测,可了解单核细胞占比变化,有助于揭示单核细胞在疾病发生和转归中的作用。鉴于小鼠单核细胞特异性抗原CD115的稳定性较差,在流式检测过程中,对其影响因素进行优化,以确保检测结果的准确性。首先,探讨CD115抗原-抗体结合前后温度和放置时间对其影响及多聚甲醛(paraformalclehyde,PFA)固定对CD115稳定性的保持作用。在此基础上,通过组合流式抗体CD11c、CD49b、Ly6G、Ter119、CD3e和B220选定阴性细胞群,再用CD11b和CD115圈门得到双阳性的单核细胞,最后根据Ly6C表达强弱区分单细胞亚群。结果显示,温度和放置时间对小鼠单核细胞CD115的稳定性影响显著。将小鼠样本在冰上放置6 h时,CD11b和CD115双阳性细胞比例仍保持稳定,在室温放置1 h即明显下降,在37℃放置0.5 h则大部分消失。该结果在抗体结合前后趋势一致。PFA固定后,在4℃条件下避光保存,CD11b和CD115双阳性细胞比例可稳定保持3 d。在此优化条件下,应用组合抗体可较好地分析小鼠单核细胞及其亚群,为精准检测小鼠单核细胞提供了技术支持,并为进一步研究单核细胞生物学特性及其在疾病中的作用提供了可能。

As the essential part of defense system,the changes of monocytes may reflect the inflammation and other disease states to some extent.Using flow cytometry to detect the surface antigens of monocyte could analize the proportion changes of monocytes and reveal the role of monocytes in the occurrence and development of diseases.In view of the poor stability of mouse monocyte specific antigen CD115,in the process of flow detection,the influencing factors were optimized to ensure the accuracy of detection results.First,the impact of temperature and time on the analysis of CD115 was measured before or after CD115 antigen-antibody binding.Next,the effect of fixative paraformaldehyde(PFA)on the CD115 stability was also examined.In addition,mouse monocytes were analyzed by negative gating with several lineage antibodies(Lin)including CD11c,CD49b,Ly6G,Ter119,CD3e and B220,followed by positive gating with CD11b and CD115 antibodies,and finally divided into monocyte subsets by the expression level of Ly6C.The results showed that temperature and storage time had significant effects on the stability of CD115.The percentage of CD11b+CD115+cells stayed constant when incubated on ice for up to 6 h.However,the detectable CD11b+CD115+cells decreased significantly if the sample were left at room temperature for 1 h and even vanished after 0.5 h if the temperature increased to 37℃.After fixed by PFA,the percentage of CD11b+CD115+cells maintain the same for at least 3 d when stored at 4℃in the dark.Based on this optimized method,using the combined antibody could better analyze mouse monocytes and their subsets,which might detect the population more accurately,and made it possible for further investigating the biological characteristics of monocytes and their effects in disease.