Monochlorobimane mcb

To evaluate intracellular ROS levels, dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma Aldrich, St Louis, MO, USA) assay was performed similarly to as previously described [51 (link)]. After cell treatments, cell culture medium was removed, and the 5 μM DCFH-DA was incubated in PBS for 30 min, at 37 °C and 5% CO₂. The cell culture plate was washed with PBS, and fluorescence of the cells was read at 485 nm (excitation) and 535 nm (emission) using the multiwall reader Appliskan (Thermo-Fisher Scientific, Vantaa, Finland). Cellular autofluorescence was subtracted as a background using the values of the wells not incubated with the probe.
Similarly, to evaluate reduced GSH levels, monochlorobimane (MCB, Sigma Aldrich, St Louis, MO, USA) assay was performed as previously reported [52 (link)]. Cell culture medium was removed, and 50 μM MCB was incubated in PBS for 30 min, at 37 °C and 5% CO₂. The cells were washed in PBS, and their fluorescence was measured at 355 nm (excitation) and 460 nm (emission).

According to the previous method [43 (link)], the ROS in chives were determined using a Zeiss LSM 800 confocal microscope (Carl Zeiss, Oberkochen, Germany). Briefly, the leaf apex of Chinese chive was incubated with 25 μM 2′, 7′-dichlorofluorescin diacetate (H₂DCFDA; Sigma-Aldrich, Saint Louis, America, http://www.sigmaaldrich.com (accessed on 25 April 2021)) for 20 min in the dark. After washing with HEPES/NaOH buffer (pH 7.5) three times, the sample was detected immediately by confocal microscope. Detection was performed by λ (excitation) = 488 nm and λ (emission) = 500–530 nm. The relative fluorescence was expressed as values relative to the control group at 0 day.
The reduced GSH content in chives was estimated following the previous method [44 (link)]. The leaf apex of Chinese chive was incubated with 50 μM monochlorobimane (MCB; Sigma-Aldrich, Saint Louis, America, http://www.sigmaaldrich.com (accessed on 25 April 2021)) for 20 min in the dark and was washed with HEPES buffer (pH 7.5) three times. Subsequently, the sample was detected immediately by a Zeiss LSM 800 confocal microscope (Carl Zeiss, Oberkochen, Germany; emission at 461 nm, excitation at 380 nm, respectively). The relative fluorescence was presented as values relative to Con at 0 day.

Reduced glutathione (GSH) is an important antioxidant compound for CNS cells, as it serves as a substrate for peroxidases and is conjugated with free radicals [13 (link)]. Here, we evaluated the protective effect of the flavonoid agathisflavone on LPS-induced cytotoxicity in primary microglia culture, through the determination of intracellular GSH. For this, the cells were seeded at a density of 3 × 10⁴/cm², and after stimulation with LPS and treatment with agathisflavone, the cells were washed three times with PBS and incubated with 400 μL of medium containing 1 mM of monochlorobimane (MCB) (Sigma Aldrich, St. Louis, MO, USA) for 30 min in the dark. L-buthionine-S-R-sulfoximine (BSO) was used at a concentration of 1 mM as an inhibitor of GSH synthesis and adopted as a positive control. After the incubation time, the cells were washed again with PBS. The detection of free glutathione was performed using a fluorescence microscope (Leica, DFC7000, Wetzlar, Germany). Three independent experiments were carried out.