关键词: 流式细胞术, 抗体滴定, 染色指数

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