Ratiometric Analysis of Fura Red by Flow Cytometry: A Technique for Monitoring Intracellular Calcium Flux in Primary Cell Subsets
Fig 4
Calcium assay to simultaneously monitor vehicle- and drug-treated cells.
Expanded human T cells were treated with vehicle (ethanol) or Gαi inhibitor, pertussis toxin (PTX) for 1 hour at 37°C. Vehicle and PTX treated cells were stained for surface antigens, loaded with 1 μM Fura Red, AM and stained with a cell viability dye. Chemokine stimulated calcium flux was monitored simultaneous in the vehicle and PTX groups by selective gating. (A) Schematic of staining protocol and data acquisition by flow cytometry. (B) Representative example of selective gating strategy to monitor live cells, singlets, CCR6+, distinguishing vehicle- and PTX-treated cells based on anti-CD3 antibodies conjugated to distinct fluorochromes (AlexaFluor700 or APC-Cy7). Numbers next to gated population indicate percentage of cells expressing antigen of interest. (C) Ratiometric analysis of Fura Red calcium dye depicted as mean Fura Red Ratio over time. Plots depict calcium flux by CCR6+ T cells in response to CCL20 (125 ng/ml), or ionomycin (5 μg/ml). Stimulus was added at 25 seconds. (D) Data from C analyzed as the proportion of cells producing a calcium signal greater than the average background signal. One representative experiment of n = 3 shown, performed with a minimum of two technical replicates.