Generation of extracellular superoxide anion (O
2-) from peritoneal macrophages was assayed as superoxide dismutase (SOD)-inhibitable reduction of
ferricytochrome C with or without stimulation with phorbol myristate acetate (
PMA) (Dvorožňáková et al., 2008 (
link)). Peritoneal cells were aseptically harvested in
RPMI 1640 (Sigma-Aldrich, Hamburg, Germany) to a final concentration of 1x10
6 cells/ml. One ml of cell suspension was adhered to each well using
24-well plates (Falcon, France) and incubated at 37
oC in 5 % CO
2 and 85 % humidity for 2 h. The reaction was carried out in 0.3 ml/well of 160 μM
ferricytochrome C (Sigma-Aldrich, Hamburg, Germany) in Earl’s balanced salt solution (EBSS) (pH 7.2). For control the reaction was blocked by 300 μg SOD/10 μl in EBSS (Sigma-Aldrich, Hamburg, Germany). 10 μl of
PMA (Sigma-Aldrich, Hamburg, Germany) in ethanol was used for the stimulation of cells for respiratory burst. Cells were incubated at 37
oC in 5 % CO
2 and 85 % humidity for 2 h and the optical density (OD) of supernatant was measured at 550 nm. The amount of O
2- produced was calculated from the difference between OD in reactions blocked by SOD and without SOD. As the resulting value, the nanomols (nmol) of produced O
2- were calculated according to the formula: nmol O
2- = (OD/6.3) x 100 and determinated for 1 mg of cell proteins.
Vargová M., Hurníková Z., Revajová V., Lauková A, & Dvorožňáková E. (2020). Probiotic Bacteria can Modulate Murine Macrophage’s Superoxide Production in Trichinella Spiralis Infection. Helminthologia, 57(3), 226-234.
