You were assigned the CD4 T cell marker during the MACsFlow

You were assigned the CD4 T cell marker during the MACs/Flow Laboratory but by mistake you added 100lt of MACs anti CD4 antibody instead of 10lt prior to labeling these cells with FITCs anti CD4 antibody. Predict the % of the CD4 +ve cells, identified by Flow-Cytometry in the Negative and Positive selection fraction. Explain your reasoning

Solution

Since excess antibodies bind at low affinity and create a bacground that clouds the results, based on the staining index the right amount of antibody should be used. Since in MACs/Flow Cytometry in the Negative selection the antibody used is against an antigen present on cells will be different from the cells of interest i.e. some other cells here in this case different from CD4 T cell marker, hence the use of 100lt of MACs anti CD4 antibody instead of 10lt will not change the negative selection readings. However in case of Positive selection fraction due to higher concentrations i.e. 100lt of MACs anti CD4 antibody instead of 10lt the signal-to-background will increase.