Question

You wish to determine the percentage of various types of thymocytes in a sample of cells from mouse thymus using flow cytometry.

You stain the sample with a FITC (green) label antibody that binds to CD3. Analysis of the stain sample indicates that 70% of the cells are stained. Based on this result, how many of the thymus cells in your sample are expressing antigen-binding receptors on their surface? Would all be expressing the same type of receptor? Explain your answer. What are the remaining unstained cells likely to be?

You then separate the CD3+ cells with a fluorescence-activated cell sorter from the other unstained cells. You restain the CD3+ cells. In this case, you use a PE( red)-label anti-CD4 antibody. Analysis of the stained CD3+ cells shows that 80% of them are stained for CD4. From this result, can you determine how many T_c (killer) cells are present in the sample? If yes, then how many T_c (killer) cells are there? If no, what additional experiments would you preform to determine the number of T_c (killer) cells that are present?

Answer 1

Flow cytometry is the laser based technique that is used in cell counting, cell sorting biomarker detection and protein engineering. This technique is mostly used in diagnosis of health disorders such as blood cancer, clinical practice, and clinical trials. The fluorophores are used as labels in flow cytometry In the given case, the fluorophore used in flow cytometry is fluorescein isothiocyanate (FITC). The FITC is bound to an antibody that specifically binds to the CD3 antigen on T-cells In humans, the T-cells mature in the thymus and the CD3 antigen are initially expressed in the cytoplasm of pro-thymocytes. The pro-thymocytes act as stem cells, which results in T-cells formation in the thymus The pro-thymocytes differentiate into common thymocytes, and then into medullary thymocytes. At medullary thymocytes stage, the CD3 antigen migrates to the cell membrane. Thus, all mature T-cell membranes are found bound to the antigein The high specificity of CD3 antigen to the T-cells and its occurrence at all stages of T- cell development allows us to use it as an immunohistochemical marker for T-cells in tissue sections In the given case, 70% of cells are stained. This is because almost all the T-cells possess CD3 antigens. Hence, all different types of T-cells will express CD3 antigens. The cells that are not labelled with CD3 specific antibodies might be the progenitor cells that have CD2 but lack CD3

The CD3 labelled cells further differentiate to form TH and Tc cells. The TH cells possess CD4, while Tc cells possess CD8 marker In the given case, the stained cells are separated from unstained cells and are further treated with PE labelled antibody specific for CD4 antigen. The results depict that the 80% of stained cells change their color, which indicate that they have CD4 on their surface. Based on this result we can depict that the percentage of Tc cells will be about 20% in the sample. This is because most of the CD3 labeled cells further differentiate to form TH and Te cells and become double positive for CD4 and CD8.