2.4. Real-time detection of intramitochondrial superoxide flux in unstimulated macrophages

Intramitochondrial superoxide flux in resting macrophages was detected by lucigenin-derived chemiluminescence (LDCL) described by us before [15]. Briefly, unstimulated macrophages (2 × 10⁵ cells) were suspended in 1 ml of air-saturated complete PBS (PBS supplemented with 0.5 mM MgCl₂, 0.7 mM CaCl₂, and 0.1% glucose). The LDCL response was initiated by adding 5 μM lucigenin, and the real-time photon emission (counts per minute) was captured continuously for 60 min at 37°C using an ultrasensitive LB9505 multichannel luminometer (Berthold, Wildbad, Germany). To determine the effects of the nanoparticles, nanoceria were added to the cell suspension immediately before the addition of lucigenin. Hence, the total incubation time with nanoceria was 60 min. Likewise, to further confirm the source of cellular superoxide, METC inhibitors, mitochondrial uncoupler (FCCP), or known superoxide scavengers were also added immediately before the addition of lucigenin. The total counts of photons emitted during the 60 min were calculated as integrated LDCL response which indicated the total amounts of mitochondrial superoxide flux in the resting macrophages during the 60-min period. It is worth noting that lucigenin at 5 μM has been shown to be a reliable probe for detecting biological superoxide in human ML-1 cell-derived macrophages [15, 17]. In these cells at resting conditions, LDCL (with 5 μM lucigenin) specifically detects intramitochondrial superoxide, and any artifact due to potential lucigenin redox cycling is completely eliminated [15, 17]. On the other hand, very high concentrations (e.g., 50–500 μM) of lucigenin commonly used in detecting biological superoxide by many investigators may cause artifacts depending on the nature of the biological systems studied [19]. Nevertheless, in addition to lucigenin, other probes (e.g., coelenterazine hcp and DEPMPO) were also used in the present study to determine cellular superoxide and the effects of nanoceria.