Fluorescence spectrophotometer lc50

Neutrophils or transfected HL-60 cells at a density of 5×10⁷ cells/ml in KRG containing 0.1% BSA were loaded with 5 µM FURA 2-AM for 30 minutes in the dark at room temperature. The cells were then diluted 1/2 in RPMI 1640 culture medium without phenol red (PAA Laboratories GmbH, Pasching, Austria) and centrifuged. Finally, the cells were washed once with KRG and resuspended in the same buffer at a density of 2×10⁷/ml. Calcium measurements were carried out in a PerkinElmer fluorescence spectrophotometer (LC50), with excitation wavelengths of 340 nm and 380 nm, an emission wavelength of 509 nm, and slit widths of 5 nm and 10 nm, respectively. The transient rise in intracellular calcium is presented as the ratio of fluorescence intensities (340 nm: 380 nm) detected. The measuring cuvette contained catalase (2000 U) to counteract inactivation of the chemoattractants by the MPO-H₂O₂-system [29] (link). The transient rise in intracellular calcium was also examined using an Accuri C6 flow cytometer (Becton Dickinson Sparks, MD, USA) with cells loaded with Fluo-3 AM (4 µg/ml) and Fura Red AM (10 µg/ml). The relative calcium level was expressed as a ratio between Fluo-3 and Fura Red (FL-1/FL-3) fluorescence over time.