Ratiometric [Ca²⁺]i measurements in adherent cell-lines using the NOVOstar microplate reader

Area of Science:

• Biomedical Science
• Cell Biology
• Analytical Chemistry

Background:

• Intracellular calcium concentration ([Ca(2+)]) is a key regulator of cellular processes.
• Accurate measurement of [Ca(2+)] is essential for medical science research.
• Existing methods may be costly or limited in throughput.

Purpose of the Study:

• To describe a method for measuring intracellular calcium concentration using the NOVOstar microplate reader.
• To provide a cost-effective and medium-throughput solution for [Ca(2+)] determination.
• To guide researchers in calibrating measurements to absolute concentrations.

Main Methods:

• Adherent cells are cultured in well plates and loaded with the fluorophore fura-2.
• Autofluorescence is measured, followed by [Ca(2+)] determination using the NOVOstar system.
• Calibration to absolute concentrations (nM) is achieved using ionomycin, EGTA, and the Grynkiewicz equation.

Main Results:

• The NOVOstar microplate reader can be configured to measure [Ca(2+)] in adherent cell lines.
• The method allows for medium-throughput analysis.
• Fluorescence data can be accurately converted to absolute intracellular calcium concentrations.

Conclusions:

• The NOVOstar microplate reader provides a versatile and affordable system for measuring intracellular calcium.
• This method facilitates the study of cellular activation and related events.
• The described protocol enables precise quantification of [Ca(2+)] for diverse research applications.