You are going to use flow cytometry to determine the proportion of developing B cells in the bone marrow that are immature, anergic, or mature. You have three monoclonal antibodies specific for three different B-cell surface proteins

Question

The first has specificity for the cell-surface protein CD19, which is expressed by all developing and mature B cells; the second is specific for the Fc region of IgD; and the third is specific for the Fc region of IgM. The antibodies are conjugated to three different fluorescent tags that can be detected and distinguished by the flow cytometer.
A. Use histograms to show your analysis of CD19-positive cells and indicate which part of your histogram you would gate to analyze IgM and IgD expression. Indicate the gated population with an arrow.
B. Using a two-dimensional dot plot, compare the expression of IgD and IgM of these gated cells, and say which of these populations represents (i) immature B cells, (ii) mature B cells, and (iii) anergic B cells.

Answer 1

A. Histogram. Your histogram should look like the one in the left panel of Figure A6.3, with axes labeled ‘cell number' (y) and ‘relative fluorescence intensity' (x) depicting total bone marrow cells stained with anti-CD19. Two peaks will be observed. One is the CD19-negative population and the other is the CD19-positive population. Gate the CD19-positive population (developing and mature B cells) for two-dimensional dot-plot analysis.
B. Two-dimensional dot plot. Your dot plot should look like the one in the right panel of Figure A6–46, with relative fluorescence intensity of anti-IgD versus anti-IgM. Three populations of B cells will be distinguished: (1) IgM-high, IgD-low, which represent immature B cells; (2) IgM-low, IgD-high, which represent mature B cells; and (3) IgM-very low (retained in the cell), IgD-high, which represent anergic B cells.