Synergy fluorescence plate reader

The fluorescent probe 2’7’-dichlorofluorescein-diacetate (DCFH-DA; Sigma-Aldrich, St. Louis, MO) was used to measure ROS production in Bge cells following a method described previously with hemocytes [33 ]. Bge cells (~1.5 x 10⁵) in suspension were washed 3X with CBSS before incubation in CBSS (control), CBSS containing either 30 μg/mL LTP, 1 mM ZnCl₂ or 30 μg/mL LTP + 1 mM ZnCl₂ for 1 hr at 26°C. After treatment, cells were washed 3X with CBSS and centrifuged at 1000 rpm for 10 min. The final cell pellets were then re-suspended in 150 μL of CBSS containing 10 μM DCFH-DA, and distributed in three wells of a 96-well black-walled plate (BD Falcon). The oxidation of DCFH-DA to fluorescent 2’7’-dichlorofluorescein (DCF) was measured in triplicate at 10 min intervals for up to 60 min using a Bio-Tek Synergy fluorescence plate reader (Winooski, VT) with excitation and emission wavelengths of 485 ± 20 and 528 ± 20, respectively. Data analysis was conducted with Origin software (Microcal, Northhampton, MA, USA). Five independent replicates of each experiment were conducted, with the raw data presented as mean ± SEM, and ratios of means of treated groups to controls presented separately.

BV-2 cells transduced with RFP-lentivirus were treated with methamphetamine in a time-dependent manner or were pretreated with BD1047 for one h and then treated with methamphetamine. The BV-2 cells were then stained for 30 min with 10 μM carboxy-H2DCFDA (Molecular Probes, Eugene, OR); 1 μM Hoechst 33342 (Molecular Probes, Eugene, OR) was added during the last 5 min of the incubation. After the incubation, the cells were washed with PBS and then immediately visualized using a fluorescence microscope (Zeiss, Göttingen, Germany) or resuspended in PBS containing 20 mM glucose before being analyzed with a Synergy fluorescence plate reader (Bio-Tek Instruments, Winooski, VT). The DCF fluorescence values were divided by the corresponding Hoechst fluorescence values for normalization.

After treatment, each well was washed once with 1 mL DPBS and then incubated for 1 h at 37 °C with 500 μL of the fluorescent probe resazurin (44 µM final concentration). Resorufin was detected at 540 nm excitation and 590 nm emission wavelengths using a Synergy fluorescence plate reader (BioTeK).