A new method of determining intracellular free Ca2+ concentration using Quin2-fluorescence

Area of Science:

• Biochemistry
• Cell Biology
• Spectroscopy

Background:

• Intracellular free calcium (Ca2+) plays a crucial role in various cellular processes.
• Accurate measurement of Ca2+ concentration is vital for understanding cell function and disease.

Purpose of the Study:

• To determine the intracellular free Ca2+ concentration in suspended mouse leukemia L1210 cells.
• To utilize fluorescence spectroscopy and time-resolved measurements for precise Ca2+ quantification.

Main Methods:

• Incorporation of 2-[(2-amino-5-methylphenoxy) methyl]-6-methoxy-8-aminoquinoline-N,N,N',N'-tetraacetic acid, tetrapotassium salt (Quin2) acetoxy methylester into L1210 cells.
• Comparison of fluorescence spectrum patterns in cell suspensions with Quin2-Ca2+ complex in buffer solution.
• Time-resolved fluorescence spectroscopy to measure fluorescence lifetimes of Quin2.

Main Results:

• Fluorescence spectrum patterns were analyzed for Quin2 in cell suspensions and buffer solutions.
• The fluorescence lifetime of Quin2 bound to Ca2+ (10 ± 1 ns) was significantly longer (4.5–11 times) than free Quin2 (1.5 ± 0.5 ns).
• Plotting the long lifetime component fraction against CaCl2 concentration allowed for Ca2+ quantification.

Conclusions:

• The intracellular free Ca2+ concentration in mouse leukemia L1210 cells was determined to be approximately 35 nM.
• This study provides a precise method for measuring intracellular Ca2+ in leukemia cells using Quin2 fluorescence.
• The findings contribute to a better understanding of calcium homeostasis in cancer cells.