How do you determine the concentration of antibody to use in your custom mass cytometry panels?

We identify the appropriate antibody concentration for markers on our panel using a combination of stain index, arcsinh ratios, and spillover consideration. The verification method that we use depends on the type of marker–phenotypic, those used to identify cell types, or functional, those used to characterize cell state. For phenotypic markers, we determine the optimal concentration using the stain index, calculating the separation between positive and negative populations to find the optimal concentration that minimizes background noise and ensures an accurate signal. In Figure A above, the stain index was calculated for Tbet at varying concentrations, and 3 µg/ml was selected as the concentration with the best separation between positive and negative populations, and with the background noise minimized.

For functional markers that don’t have a strong separation between positive and negative populations, we use the arcsinh ratio to compare antibody staining in for a marker before and after stimulation. To calculate the arcsinh ratio, we arcsinh-transform the median intensity values (to make them easier to compare) and look at the difference between these transformed values for stimulated and unstimulated conditions. In Figure B above, the unstimulated (on the left) and stimulated (on the right) staining conditions are shown. The arcsinh ratio was then calculated for selected concentrations and 3 µg/ml was selected as the optimal concentration.

Additionally, if the separation of the positive signal of a custom marker is not clear and may have spillover from another channel, we also assess for spillover using a “mass minus one” (MMO) control, where we omit one marker at a time to see if it appears in a channel where it shouldn’t. We then choose the antibody concentration that provides a strong signal in the intended channel with minimal background staining in other channels.

Through these methods, we establish the optimal concentration for each marker on the panel and subsequently validate it on control samples, comparing our results with reported flow cytometry data to ensure consistency and reliability.