Summary results of AO testing of neutrophil and epithelial cell components in the enhanced chemiluminescence AO assay
| Component tested | Quantity, dilution, and/or volume | Measurable AO activity (% loss caused by NEM) | Recovery curve profile |
|---|---|---|---|
| Quantity refers to nM added to assay; for FMLP stimulated PMNL and gingival epithelial cells see materials and methods for details of original cell numbers, etc; 100 μl of GCF eluate is equivalent to an entire single GCF sample; GCF filtrates were produced by passage through 30, 10, or 3 kDa Microcon concentrating filters. *Assayed with the addition of 5 μl of 20% serum; †complete light suppression without recovery. | |||
| AO, antioxidant; GCF, gingival crevicular fluid; FMLP, F-Met-Leu-Phe; NEM, N-ethylmaleimide; PMNL, polymorphonuclear leucocytes. | |||
| FMLP stimulated PMNL* | |||
| Aerobic | |||
| Supernatant | 5–20 μl | – | – |
| Pellet | 5–20 μl | – | – |
| Anaerobic | |||
| Supernatant | 5–20 μl | + | Stepped |
| Pellet | 5–20 μl | – | – |
| Intracellular reducing agents | |||
| Taurine | 1–30 nM | –† | None |
| Hypotaurine | 0.24 nM | –† | None |
| β NADP* | 0.66 nM | – | – |
| β NADPH* | 0.66 nM | + | Stepped |
| Cysteamine* | |||
| –NEM | 2 nM | + | Stepped |
| +NEM | 2 nM | + (80%) | Smooth |
| L cysteine* | |||
| –NEM | 1 nM | + | Stepped |
| +NEM | 1 nM | + (76%) | Smooth |
| Oxidised glutathione* | 1 nM | – | – |
| Reduced glutathione* | |||
| –NEM | 1 nM | + | Stepped |
| +NEM | 1 nM | + (80%) | Smooth |
| Gingival epithelial cells* | |||
| Supernatant | |||
| –NEM | 5–20μl | + | Stepped |
| +NEM | 5–20 μl | + (78–87%) | Smooth |
| Pellet | 5–20 μl | – | – |
| GCF | |||
| –NEM | 100 μl eluate | + | Stepped |
| +NEM | 100 μl eluate | + (75–80%) | Smooth |
| GCF filtrate | |||
| <30 kDa | 100 μl | + | Stepped |
| <10 kDa | 100 μl | + | Stepped |
| <3 kDa | 100 μl | + | Stepped |
| Plasma | 1/5–1/500 (20 μl) | + | Smooth |
| Serum | 1/5–1/500 (20 μl) | + | Smooth |