Table 3.

Design of experiments on light emission from Fe²⁺-EGTA-H₂O₂ system.

No	Sample	Volumes of Working Solutions Added to Luminometer Tube (µL)
		A	B *	C **	D	E
		PBS	EGTA	FeSO4	H2O2	H2O
1	Complete system	940	20	20	100	-
2	Incomplete system I	960	-	20	100	-
3	Incomplete system II	960	20	-	100	-
4	H2O2 alone	980	-	-	100	-
Additional controls
5	Fe2+-EGTA without H2O2	940	20	20	-	100
6	Medium alone	980	-	-	-	100

Working solutions were mixed in alphabetical order. A—sterile phosphate buffered saline (PBS) (pH = 7.4) without divalent cations; B—10 mmol/L aqueous solution of EGTA; C—5 mmol/L aqueous solution of FeSO₄. Then after gentle mixing the tube was placed into luminometer chain, incubated for 10 min at 37 °C and then 28 mmol/L H₂O₂ (D) or water (E) was automatically injected with dispenser and total light emission was measured for 2 min. * in certain experiments the same concentration of EDTA or citric acid solution was added instead of EGTA solution. ** in certain experiments the same concentration of CuSO₄, CoSO₄, MnSO₄ or CrCl₂ solution was added instead of FeSO₄ solution. Some experiments were performed with 2- and 5-times lower and 2-times higher concentrations of FeSO₄, EGTA and H₂O₂.