A flow-cytometric method for continuous measurement of intracellular Ca(2+) concentration

Area of Science:

• Cellular Biology
• Biophysics
• Neuroscience

Background:

• Intracellular calcium (Ca2+) concentration changes rapidly in response to stimuli, especially in the nervous system (nanosecond timescale).
• Accurate measurement of these rapid Ca2+ dynamics is challenging.
• Existing methods like confocal microscopy and spectrofluorometry have limitations in cell throughput and temporal resolution.

Purpose of the Study:

• To evaluate a new generation of flow cytometers for dynamic intracellular Ca2+ measurements.
• To demonstrate the capability of continuous monitoring of thousands of cells for Ca2+ analysis.

Main Methods:

• Utilized an Accuri C6 flow cytometer for continuous monitoring of intracellular Ca2+.
• Employed Ca2+ modulating agents: thapsigargin (TG) to induce Ca2+ release from the endoplasmic reticulum (ER) and 2-aminoethoxydiphenyl borate (2-APB) to partially inhibit store-operated Ca2+ channels (SOCC).
• Performed calibration using standard curves.

Main Results:

• Successfully monitored dynamic Ca2+ concentration changes in real-time.
• Observed a significant increase in intracellular Ca2+ with TG, followed by partial inhibition with 2-APB, consistent with known Ca2+ signaling pathways.
• Demonstrated the ability to analyze Ca2+ responses in thousands of cells simultaneously.

Conclusions:

• Next-generation flow cytometry offers an accessible and accurate method for dynamic Ca2+ concentration measurements.
• This technique provides extensive data on cellular responsiveness and population health.
• Enables detailed analysis of rapid cellular signaling events.