Table 1.
Optimized chemiluminescence buffer composition to detect low levels of ROS.
| Componenta | Figure 1 | Figure 2 | Figure 3 | Figure 4 |
|---|---|---|---|---|
| Luminolb | 25-200 μM | 50 μM | 50 μM | 50 μM |
| 4IPBAc | 180 μM | 180 μM | 180 μM | 180 μM |
| HRPd | 0.1 or 0.2 units/ml | 0.2 units/ml | 0.2 units/ml | 0.2 units/ml |
| H2O2 e | 1-10 μM | 1-10 μM | — | |
| PMA | — | — | 200 nM | 200 nM |
| Cellsf | — | — | 2 × 105 | 2 × 105 cells in HBSS |
| Supernatants | — | — | — | 180 μl from Opti-MEM (1.2 × 106 cells) |
| Bufferg | HBSS | HBSS | HBSS | HBSS, Opti-MEM |
aFinal volume of the reaction was 200 μl in a glass bottom 96-well plate. bLuminol was dissolved in 100 mM NaOH or DMSO. Previous studies used 200 μM or 1.25 mM luminol in DMSO [9, 13] or 50 μM luminol dissolved in 100 mM NaOH [8]. c4IPBA was dissolved in DMSO. A previous study used 200 μM [13]. dPrevious studies used 0.32 unit/ml or 50 units of HRP [8, 9]- or HRP-conjugated antibody [13]. eA previous study used 2.4 mM H2O2 [13]. fPrevious studies used 5 × 105 or 102-106 cells [8, 9]. gPrevious studies used Hank's Balanced Salt Solution (HBSS), Krebs-Ringer bicarbonate buffer, or Tris buffer [8, 9, 13].