Cell preparations were suspended in 1 ml loading buffer (DPBS supplemented with 1 mM CaCl2, 1 mM MgSO4, and 1% FBS) at a density of 1 × 107 cells per ml and loaded with the calcium indicator dyes Fluo-4 (Thermo Fisher Scientific) and FuraRed (Thermo Fisher Scientific) at 37°C according to manufacturer’s directions. Cells were then washed and stained for flow cytometry at room temperature. Immediately before measurement, cells were incubated at 37°C for 5 min and then transferred to the flow cytometer for continuous monitoring. After obtaining a baseline measurement for 30 s, anti-CD3 antibodies (5 μg/ml) and IgG crosslinking antibodies (25 μg/ml) were added to the sample. After 180 s, ionomycin (1 μg/ml) was added to stimulate maximal cytosolic calcium influx.
Protocol detail
Calcium Influx Monitoring in Activated T Cells
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Corresponding Organization : Yale University
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Variable analysis
independent variables
- Anti-CD3 antibodies (5 μg/ml)
- IgG crosslinking antibodies (25 μg/ml)
- Ionomycin (1 μg/ml)
- Cytosolic calcium influx
- Cell density (1 × 10^7 cells per ml)
- Loading buffer (DPBS supplemented with 1 mM CaCl2, 1 mM MgSO4, and 1% FBS)
- Calcium indicator dyes (Fluo-4 and FuraRed)
- Incubation temperature (37°C)
- Staining and measurement at room temperature
- Baseline measurement for 30 s before stimulation
Annotations
Based on most similar protocols
Optimize cell density for loading with calcium indicator dyes Fluo-4 and FuraRed. The Input protocol uses 1 × 10^7 cells per ml, which may need to be adjusted based on cell type and experimental needs (Protocol 3 suggests 5 × 10^6 cells/ml).
Include a positive control to validate the calcium signaling response, such as adding ionomycin to induce maximal cytosolic calcium influx (Input protocol, Protocol 2, Protocol 5).
Monitor baseline calcium levels for 30 seconds before stimulation to establish a reference point (Input protocol, Protocol 2, Protocol 5).
Incubate cells at 37°C for 5 minutes immediately before measurement to ensure optimal dye loading and cell function (Input protocol).
Use appropriate isotype controls for antibody staining to account for non-specific binding (Protocol 1, Protocol 5).
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