Calcium Signalling Analysis App

This application, deployed on ShinyApps.io, is a bespoke tool developed to streamline and automate the routine data analysis tasks for calcium signalling experiments performed by the Immception Lab at INSERM UMR1291.

It is designed to handle calcium flux assay's, time-series fluorescence measurements and provide immediate visualization and qualification of cellular responses.

✨ Key Features

The app automates the standard analytical pipeline, offering flexible control over key experimental parameters:

Feature	Description
Flexible Time Windows	Users can dynamically define the start and end frame indices for Baseline, HBSS (Wash), Agonist, and Ionomycin phases, ensuring the tool is adaptable to new experimental protocols.
Robust Data Processing	Automatically processes multiple .csv files simultaneously, performing per-cell Fold Change (FC) calculation based on the user-defined Baseline.
Cell Quality Control	Implements automated filtering to qualify "Living Cells" (Ionomycin-responsive) and exclude unstable traces (HBSS-responsive), based on user-defined FC thresholds.
Mean Trace Visualization	Dynamic Plotting: Displays the Mean Fold Change over time (± Standard Error across replicates) for selected cell lines and doses, with dynamic background shading and vertical lines marking experimental phases.
Individual Traces (QC)	Provides a plot of sampled individual cell traces for quick quality control and visual assessment, ensuring high rendering efficiency for interactive use.
Quantitative Analysis	Calculates and visualizes key metrics, including Mean Max Agonist FC, Percentage of Responding Cells, and Area Under the Curve (AUC) during the Agonist phase.

🚀 Getting Started

Data Format

For successful processing, ensure your raw .csv files adhere strictly to the following naming convention and structure:

1. Filename: CellLine_Dose_Replicate.csv
   • Example: HEK293WT_8uM_1.csv, HEK293HA-E_16uM_2.csv
2. File Structure:
   • Column 1: Time point (or Frame Index).
   • Subsequent Columns: Tab-separated measurements of fluorescence intensity for each individual cell.

Usage Instructions

1. Access the App: Open the application on the shinyapps.io.
2. Upload or Demo: Use the "Browse" button to upload multiple .csv files, or click "Load Demo Data" for an immediate test of the application's capabilities.
3. Set Parameters: Adjust the Define Time Windows (Frame Index) and Define Response Thresholds (Fold Change) to match the specific kinetics and quality controls required for your current experiment.
4. Visualize Results: Navigate through the tabs to review mean traces, sampled individual cell data, bar charts for Max FC and AUC, and the final results table.
5. Download: Use the dedicated Download PNG/PDF buttons in each tab to export publication-ready figures, and the Download Table (.csv) button to export the processed per-cell data.

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💻 Technical Stack

• Platform: R Shiny, deployed on shinyapps.io
• Core Libraries: data.table (for high-performance data processing), future and promises (for asynchronous file loading), ggplot2 and plotly (for dynamic visualization).
• Developed by: Rüçhan Ekren, PhD, Bioinformatics Data Scientist
• For: Immception Lab members at INSERM UMR1291, Toulouse, France.