Barcoding T Cell Calcium Response Diversity with Methods for Automated and Accurate Analysis of Cell Signals (MAAACS)

Abstract

We introduce a series of experimental procedures enabling sensitive calcium monitoring in T cell populations by confocal video-microscopy. Tracking and post-acquisition analysis was performed using Methods for Automated and Accurate Analysis of Cell Signals (MAAACS), a fully customized program that associates a high throughput tracking algorithm, an intuitive reconnection routine and a statistical platform to provide, at a glance, the calcium barcode of a population of individual T-cells. Combined with a sensitive calcium probe, this method allowed us to unravel the heterogeneity in shape and intensity of the calcium response in T cell populations and especially in naive T cells, which display intracellular calcium oscillations upon stimulation by antigen presenting cells.

CalQuo 2 : Automated Fourier-space, population-level quantification of global intracellular calcium responses

“…Additionally, the Ca 2+ intensity amplitude and Ca 2+ intensity peak width is also provided by CalQuo 2 (see Supplementary Information). By analyzing the response function in Fourier space, CalQuo 2 is robust against noise in the raw data, offering improved performance over algorithms based on a single threshold 21 .…”

A Markovian Entropy Measure for the Analysis of Calcium Activity Time Series

“…Recently developed highly sophisticated feature-based methods can take fluorescent image files of populations of cells as their inputs, and return statistical analyses that incorporate both spatial and temporal information from the experimental measurements into their assessments of calcium dynamics [12, 35, 36]. Our Markovian Information Entropy method is currently restricted to the subset of the analysis process which converts a calcium activity time series into a single numerical value for downstream analysis, and so any upstream image processing or downstream statistical analysis must be conceived and performed by the experimenter.…”

The functional contribution of calcium ion flux heterogeneity in T cells

“…The ability to continuously track single cell's Ca 2+ responses over time is advantageous over flow cytometry, and has allowed a greater appreciation of Ca 2+ ‐dependent pathways 75 , 76 . We and others have shown that flow cytometry is unable to decipher the extent of activation, as calculated by the frequency of responding cells that are contributing to the overall response 75 , 77 , 78 . By using single‐cell analysis coupled to fluorescence microscopy, the information encoded in heterogeneous oscillations can be expressed by several parameters such as those observed by flow cytometry, including mean, peak concentration or fluorescence ratio and the change in fluorescence ratio 79 , 80 , 81 , 82 .…”