How do you test and select an optimal antibody concentration to stain intranuclear markers by mass cytometry?

Each antibody on Teiko’s cytometry panels goes through a multi-step verification process to ensure that the selected concentration gives the optimal marker detection without spilling into neighboring channels. The process for any marker starts with a six-point concentration series, beginning at 6 ug/mL and performing two-fold dilutions down to 0.1875 ug/mL to create six distinct concentrations.

For each concentration, we measure the median channel values (such as median fluorescence intensity in flow cytometry) and the standard deviation of the channel values for each marker in the cell populations that express (positive population) and do not express (negative population) the given marker. These measurements allow us to calculate a Staining Index (SI) for each concentration. The SI is a key metric in cytometry, indicating the antibody’s ability to distinguish between positive and negative populations.

When comparing staining indices, the goal is to select the concentration with the highest staining index. However, when staining indices are similar, we also look at the visual separation between positive and negative populations using the dot plot on the gating scheme, spillover into the +1, -1, and +16 channels, and the median channel value of the positive population at each concentration. A +1 channel refers to the isotope with a molecular weight one unit higher than the metal isotope bound to the antibody, while a -1 channel refers to the isotope one unit lower. We check the +16 channel to ensure the metal has not undergone oxidation.

Click below to see an example of how we verified the optimal concentration of Tbet, an intranuclear (transcription factor) marker expressed on CD4+ helper T cells, on our panel.

Tbet Example