Abstract: Flow cytometry is one of the important methods to study and diagnose hematological diseases, which can realize high-speed quantitative analysis and sorting distinct cell populations. However, due to different cells and different experimental conditions used in different experiments, non-specific staining of antigen-negative cells often occurs. Using antibody titration method, the experimental conditions for the optimal separation of antigen positive cell population and negative cell population can be obtained by calculating and comparing staining indexes. In order to optimize the experimental conditions for staining with fluorescent dye conjugated antibodies in flow cytometry, mouse bone marrow cells were taken as labeled cells, and Rat Anti-Mouse CD11b antibody labeled with non-tandem fluorescent dye FITC (FITC Rat Anti-Mouse CD11b) and Rat Anti-Mouse CD11b antibody labeled with tandem fluorescent dye APC-efluor 780 (APC-eFluor780 Rat Anti-Mouse CD11b) were selected for labeling. By calculating the staining index of mouse bone marrow cells labeled with serially diluted antibodies, antibody titration was carried out to determine the appropriate antibody concentration interval, and then the effects of cell number, staining time and fixing steps on antibody staining index were analyzed to explore the key factors affecting antibody staining of blood cells. The results showed that the staining index of FITC Rat Anti-Mouse CD11b and APC-eFluor780 Rat Anti-Mouse CD11b were higher in the range of 0.156~2.500 μg·mL-1 and 0.25~1.00 μg·mL-1, respectively, but the antibody concentration beyond this range would reduce the staining index. When the antibody concentration and staining time were constant, the FITC Rat Anti-Mouse CD11b and APC-eFluor780 Rat Anti-Mouse CD11b have higher staining index in the range of 1.56×105~5.00×106 cells·tube-1 and 1.56×105~3.12×105 cells·tube-1 respectively, but the number of cells beyond this range would reduce the staining index. When the concentration of antibody and the number of cells were constant, the staining index of FITC Rat Anti-Mouse CD11b decreased with the extension of staining time, while APC-eFluor780 Rat Anti-Mouse CD11b was opposite. By comparing the staining indexes before and after fixation, it was found that the staining indexes of FITC Rat Anti-Mouse CD11b and APC-eFluor780 Rat Anti-Mouse CD11b both decreased significantly after fixation (P<0.01 and P<0.05). The study provided a method for optimizing flow analysis of blood cells through antibody titration, and pointed out that selecting appropriate antibody concentration, cell number, staining time and fixing steps according to antibody titration results in specific experiments was of great importance to the study of flow detection of labeled blood cells.

Key words: flow cytometry, antibody titration, staining index

摘要： 流式细胞术（flow cytometry）可以实现高速、逐一的细胞定量分析和分选，是研究和诊断血液病的重要手段之一。但是由于不同实验所用细胞和实验条件不同，经常存在抗原阴性细胞非特异染色等问题。利用抗体滴定法，可通过计算、比较染色指数，得到使抗原阳性细胞群和阴性细胞群达到最佳分离效果的实验条件。为了优化血液细胞流式细胞术中荧光抗体染色的实验条件，以小鼠骨髓细胞为被标记细胞，选择利用非串联荧光染料FITC标记的大鼠抗小鼠CD11b抗体（FITC Rat Anti-Mouse CD11b）和串联荧光染料APC-eFluor780标记的大鼠抗小鼠CD11b抗体（APC-eFluor780 Rat Anti-Mouse CD11b）进行标记。通过计算不同浓度抗体标记小鼠骨髓细胞的染色指数进行抗体滴定，确定合适的抗体浓度区间，进而分析细胞数量、染色时间及固定步骤对抗体染色指数的影响，探究影响血液细胞抗体染色的关键因素。结果显示，FITC Rat Anti-Mouse CD11b和APC-eFluor780 Rat Anti-Mouse CD11b的浓度分别在0.156~2.500 μg·mL-1和0.25~1.00 μg·mL-1范围内染色指数较高，但是超出这个范围的抗体浓度会使染色指数降低；抗体浓度、染色时间一定时，FITC Rat Anti-Mouse CD11b和APC-eFluor780 Rat Anti-Mouse CD11b分别在细胞数量为1.56×105~5.00×106 cells·管-1和1.56×105~3.12×105 cells·管-1范围内染色指数较高，但是超出这个范围的细胞数量会使染色指数降低；抗体浓度、细胞数量一定时，对于FITC Rat Anti-Mouse CD11b，随着染色时间的延长，染色指数降低，而APC-eFluor780 Rat Anti-Mouse CD11b与之相反；通过比较固定前后染色指数的高低发现，FITC Rat Anti-Mouse CD11b和APC-eFluor780 Rat Anti-Mouse CD11b在固定后染色指数均显著下降（P<0.01和P<0.05）。研究结果提供了一种通过抗体滴定优化流式分析血液细胞的方法，并指出在特定实验中根据抗体滴定结果选择合适的抗体浓度、细胞数量、染色时间和固定步骤对标记血液细胞进行流式检测的研究至关重要。

关键词: 流式细胞术, 抗体滴定, 染色指数