关键词: 单核细胞, 流式细胞术, CD115

Abstract: 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.

Key words: monocyte, flow cytometry, CD115