and lucigenin levels, bcl-2/bax ratio, [Na.sup.+]/[K.sup.+]-ATPase, caspase-3, and caspase-9 activities of the frozen samples were examined once thawed at the room temperature.
Antioxidant Capacity Assay by Luminol
It is based on CR1/CR3 receptor activation of neutrophils in the whole blood, which produce oxidants that react with luminol
The ROS levels in the rat retina were analyzed using luminol
(5-amino-2,3-dihydro-1,4-phthalazinedione, Sigma) and lucigenin (bis-N-methylacridiniumnitrate, Sigma).
Briefly, the following were added into 850 [micro]L of Hanks' balanced salt solution (HBSS) (pH 7.4): 10 [micro]L of luminol
(1 mM), [H.sub.2][O.sub.2] (100 mM), sodium selenite (IV) (2mM), and CoCl2 x 6H2O (II) (1 mM).
50 [micro]L of luminol
(0.28 mM) and 50 [micro]L of different concentrations of the propolis and bud poplar resins extracts were added to 100 [micro]L of neutrophil solution (1.25 x [10.sup.6] cells/mL) and the mixture was incubated for 3 min at 37[degrees]C.
Briefly, using the chemiluminescence technique to measure the antioxidant capacity of biological fluids, the light emission occurs when the chemiluminescent substrate (i.e., luminol
) is oxidized by [H.sub.2][O.sub.2] in a reaction catalyzed by "horseradish peroxidase." The stabilities and intensities of the light emitted are high due to the addition of p-iodine phenol.
Katsu, "Porphyrin-induced photogeneration of hydrogen peroxide determined using the luminol
chemiluminescence method in aqueous solution: a structure-activity relationship study related to the aggregation of porphyrin," Analytical Sciences, vol.
Soybean lecithin (Calbiochem), K[H.sub.2]P[O.sub.4], [Na.sup.2]HP[O.sub.4], peptone, HCl, [H.sup.2][O.sup.2], luminol
(5-amino-2, 3-dihydrophthalazine-1, 4-dione), and Tris (hydroxymethyl aminomethane base) were purchased from Merck (Germany).
Briefly, the macrophages were adhered to round glass coverslips (9 mm) at concentration of 1 x [10.sup.5] cells per coverslip and then placed in cuvettes containing 1 ml of Hank's solution (luminol
0.8 x [10.sup.-4]M and HRP at 4U).
The most common method for detecting blood stains is to spray a crime scene with luminol
, a chemical that reacts with iron in blood to emit a blue glow that can be seen in the dark; the use of luminol
has its disadvantages.
Camptothecin (98% purity), luminol
, f-MLP (formyl-met-leuphenylalanine), SAAVNA (N-succinyl-alanine-alanine-valinine-pnitroanilide), cytochalasin B, TMB (3,3',5, 5'-tetramethylbenzidine) liquid substrate system, Hanks' balanced salt solution (HSSB), L-glutamine, fetal bovine serum (FBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and RPMI1640 medium were purchased from Sigma-Aldrich GmbH (Steinheim, Germany).