Flow cytometric study of changes in the intracellular free calcium during the cell cycle

We measured the intracellular levels of free cytoplasmic calcium in different phases of the cell cycle in viable rat fibroblasts, using two parameter flow cytometric analysis with Hoechst 33342 as the DNA specific dye and Fluo-3 as the calcium sensitive dye. We studied changes in calcium levels during the G1 phase of cell cycle by arresting cells with chemical agents such as staurosporine and hydroxyurea or by density dependent arrest of cell growth. We show that levels of calcium are lowest at the beginning of G1 phase but rise steadily with its progression and culminate at the G1/S border. Our results suggest that complex changes occur in calcium levels during the process of mitotic division. During progression of S and G2 phases, calcium levels decline and increase respectively. Our results offer a new methodology to estimate intracellular calcium levels in specific phases of the cell cycle. Based upon these results we propose a general scheme representing the changes in the intracellular calcium concentration during the progression of the cell cycle.