A Quantitative Method to Measure Low Levels of ROS in Nonphagocytic Cells by Using a Chemiluminescent Imaging System

Table 1

Optimized chemiluminescence buffer composition to detect low levels of ROS.

Component^a	Figure 1	Figure 2	Figure 3	Figure 4
Luminol^b	25-200 μM	50 μM	50 μM	50 μM
4IPBA^c	180 μM	180 μM	180 μM	180 μM
HRP^d	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
Cells^f	—	—		cells in HBSS
Supernatants	—	—	—	180 μl from Opti-MEM ( cells)
Buffer^g	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 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].