Zymosan phrodo green

To assess the effect of melatonin on the phagocytes, reactive oxygen species, viability, and intracellular Ca²⁺ release, mononuclear (MN)phagocytes were induced into an inflammatory process via bioparticles of Zymosan (from Saccharomyces cerevisiae). The MN cells and Zymosan were incubated for 2 h at 37 °C and treated with 199 medium (Gibco, Grand Island, NE, USA; negative control) and melatonin (Sigma, St Louis, MO, USA; final concentration 100 ng/mL). The phagocytosis assays were performed with Zymosan pHrodo Green^® (Thermo Fisher, Carlsbad, CA, USA). Free radical release, intracellular calcium, and apoptosis assays were performed with Zymosan (Sigma, St Louis, MO, USA) without conjugated fluorochrome to avoid interference in the fluorescence intensity with the other reagents.

The phagocytosis assay using Zymosan pHrodo Green™ (Thermo Fisher, Carlsbad, CA, USA) did not require wash steps and quencher dyes. So, after the incubation period, 10,000 cells were analyzed by flow cytometry using FACSCalibur^™ (BD Biosciences, San Jose, CA, USA) with excitation/emission maxima of 509/533 nm. The results were expressed by the Phagocytosis Index (%). The experiments were performed in duplicate.

The activation of mononuclear (MN) cells was performed by incubation with Zymosan in the presence and absence of the exogenous adipokines human adiponectin (Sigma, St Louis, MO, USA) and human leptin (Thermo Fisher, Carlsbad, CA, USA), each at a concentration of 100 ng/mL. The concentrations were in accordance with data from the scientific literature [25 (link)], and preliminary pilot tests were conducted to standardize the concentrations used.
The MN cells were incubated with Zymosan (for 2 h at 37 °C under gentle shaking) and treated with 199 medium (negative control), adiponectin, leptin, and adiponectin+leptin.
The phagocytosis assays were performed with Zymosan pHrodo Green™ (Thermo Fisher, Carlsbad, CA, USA), because it emits green fluorescence in the presence of an acidic pH during the phagocytosis process. Free radical release, apoptosis, and intracellular calcium assays were performed with Zymosan (Sigma, St Louis, MO, USA) without conjugated fluorochrome to avoid interference in the fluorescence intensity of the reagents used in each assay.