In any flow cytometry experiment, it is important to maximize the fluorescence signal while having low levels of background. Optimizing the separation of positive and negative signals will help to determine the optimal concentration of antibody-fluorophore conjugate needed for accurately labeling the cells of interest. In order to accomplish this, titration of all antibody conjugates used in the panel is required [4,5]. Titration allows optimization of antibody concentration that is necessary to maximize the separation between positive and negative fluorescence peaks, minimize nonspecific binding, and establish the highest labeling efficiency of an antibody.
Regularly, vendors will recommend a concentration to use based on their standard protocols. However, to obtain the best results, it is best to optimize the concentration of antibody used for specific assay conditions, including cell type and instrument used. Typically, the optimal concentration of an antibody to use in an experiment is determined by having a high stain index (SI). SI is calculated by subtracting the median fluorescence intensity (MFI) of the negative peak from the MFI of the positive peak and dividing by two times the standard deviation of the negative peak. The optimal antibody concentration will have the greatest separation between positive and negative MFI peaks. The SI also accounts for the spread of the negative population, which can decrease the separation of positive and negative populations. To demonstrate the importance of optimizing antibody concentrations for an experiment, we show results of the full staining panel with vendor- recommended antibody concentrations and with optimized antibody concentrations, and compare the results.
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As an example, isolated peripheral blood mononuclear cells (PBMCs) from healthy donors were stained with varying concentrations of an Invitrogen anti-CD3 APC-Cy®7 conjugate. SI, MFI, and percent positive cells were calculated from singlet cells as identified through a forward height vs. forward area dual-parameter plot that was then gated on live CD3-positive cells and graphed using GraphPad Prism™ software version 6.07 (Figure 1A). The optimal concentration of antibody to use is determined by the intersection of all three dotted lines that represent all three parameters. There can be a number of optimal concentrations of antibody. For example, CD3 APC-Cy7 antibody conjugate at 4.0 µg, 2.0 µg, and 1.0 µg per test can be thought to have ideal characteristics for use in a panel because the SI, MFI, and percent positive cells are at their highest level (outlined in the blue dotted box in Figure 1A). However, the optimal concentration of antibody is the lowest amount of antibody needed to sufficiently detect all positive targets within a sample, obtain a high mean fluorescent signal, and have low levels of nonspecific binding. As shown in Figure 1B, the percentage of cells is increased in the negative fluorescence peak at 4.0 µg CD3 antibody conjugate, as compared to 2.0 µg and 1.0 µg CD3 antibody conjugate per test. As in all cases, the optimal concentration of antibody is not only based on SI, MFI, and percent positive cells, but it is also dependent on low levels of background.
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