Work

Fura-10, unlike fura-2, is suitable for long-term calcium imaging in natural killer (NK) cells without compromising cytotoxicity and can be combined with target cell death analysis

Year: 2025

Type: article

Abstract: • Fura-10 does not compromise NK cell cytotoxicity against cancer cells • Fura-2 may compromise NK cell cytotoxicity against cancer cells • Fura-10 is bright, has a good signal-to-noise ratio, shows little bleaching/leaking • Fura-10 works well in primary human NK cells and murine pancreatic islets • Correlation of fura-10 NK cell’s Ca 2+ signals and cytotoxicity from cancer patients There are compelling reasons to opt for primary human natural killer (NK) cells when validating Ca 2+ indicators. 1.) NK cells exhibit a high degree of vulnerability to stressors such as indicator loading or light exposure. 2.) The lack of research on NK Ca 2+ signaling underscores the necessity for developing reliable assays. 3.) The increased utilization of NK cell therapies necessitates a more profound comprehension of Ca 2+ dependent signal transduction. Consequently, an assay was developed to monitor cytosolic Ca 2+ signals in individual NK cells simultaneously with their cytotoxic function against cancer cells. We used this assay to assess the suitability of fura-2, fura-PE3, fura-8, fura-10 or fura-red for quantifying Ca 2+ signals in NK cells without compromising their cytotoxic function. In contrast to the widely used fura-2, its red-shifted derivative fura-10 did not interfere with NK cytotoxicity over several hours. It exhibited a superior signal-to-noise ratio and good dynamic range, accompanied by minimal bleaching or leakage. Fura-8 and fura-red also preserved NK cell cytotoxicity, but had other disadvantages compared to fura-10. We successfully used fura-10 to report Ca 2+ signals in NK cells from blood donors and patients diagnosed with lymphoma and leukemia over several hours at 37°C during apoptotic or necrotic killing of different cancer cells (K562, THP1, OCI-AML2, and TMD8). Additionally, we show that fura-10 is well suited to report Ca 2+ signals in intact murine pancreatic islets, another stress-sensitive cell preparation. Consequently, fura-10 is an optimal choice for measuring Ca²⁺ in primary human NK cells and other primary cell preparations.

Source: Cell Calcium

DOI: 10.1016/j.ceca.2025.103091