NO is a key cellular messenger important to animals and plants alike. In humans, abnormal NO production is implicated in numerous pathological conditions, including circulatory diseases, cancer, inflammation, asthma, stroke, and diabetes. In the vasculature, endothelial cells (ECs) produce NO to regulate blood pressure, but drugs and environmental toxicants may impair NO production leading to cardiovascular disease (CVD), the primary cause of death in the U.S. and worldwide. Activated macrophages produce large quantities of NO, which kills pathogens but can also damage the body’s own cells (e.g., in rheumatoid arthritis). Elevated NO production is associated with tumor aggressiveness and resistance to cancer immunotherapy.
We offer a unique service for measuring NO production in cells using novel highly specific fluorescent NO probe NO530. Figure 1 presents a side-by-side comparison of NO530 and DAF images and quantitation data demonstrating the clear superiority of NO530 vs. DAF-FM DA, one of the most cited probes for NO studies. Unlike DAF staining, NO530 fluorescence in activated Raw 264.7 cells was completely inhibited by preincubation with NMMA, a competitive inhibitor of NO synthase.
Figure 1. Comparison of the effect of specific NO inhibitors on NO530 and DAF staining in RAW264.7. Cells were incubated overnight with LPS+IFN-γ, as described earlier. NMMA, a biopterin synthesis inhibitor DAHP, or vehicle control were added to cells for 1 hr and then NO530 or DAF were added for the last 15 min of pre-incubation with NMMA and DAHP. 1 mM L-arginine was added for 30 min. Images were taken with the high content imager INCA 2200 (GE Healthcare) at 20x magnification and quantified using commercial software. A) NO530 (upper row) and DAF (lower row) staining, B) quantitation of NO530 staining, and C) quantitation of DAF staining. Vehicle only – open bars, 2.5 mM NMMA – black bars, 1 mM DAHP – gray bars. Mean ± SD (n=3).
NO530 also provides excellent concentration-response to a wide range of L-arginine concentrations (Figure 2A, black bars). Pre-incubation with NMMA blocks the NO530 signal (Figure 2A, gray bars). In parallel, DAF reagent could not generate a concentration-response curve (Figure 2B). Moreover, a pre-incubation with NMMA had little effect on DAF fluorescence (Figure 2B, gray bars).
Figure 2. NO staining quantitation in RAW264.7. Cells were incubated overnight with LPS+IFN-γ. NMMA or vehicle control were added to cells for 1 hr. Then NO530 or DAF were added for the last 15 min of pre-incubation with NMMA and then L-arginine titers were added for 30 min. Images were taken with the high content imager INCA 2200 (GE) at 20x magnification and quantified using manufacturer’s software. A) NO530 staining, B) DAF-FM DA staining. Vehicle only - black bars, 2.5 mM NMMA -gray bars. Mean ± SD (n=3).