Synergy mx monochromator based multi mode microplate reader

Hormonal responses were measured 2 h after the repeated administration of CP-101,606. The levels of hormones in serum or pituitary were measured using ELISA kits for GHRH, GH, corticosterone and thyroid hormones (T₃, T₄). The serum concentrations of cytokine were analyzed using the IL-6 ELISA kits. Absorbance was measured using a Synergy Mx Monochromator-Based Multi-Mode Microplate Reader (Biotek, Winooski, VT, USA).
The hormone and cytokine concentrations were calculated from 8–10 rats for the control and drug-treated group in the 5-day experiment or from 11–13 animals for the control and CP-101,606-treated group in the 3-week study.

Acridine orange is a versatile fluorescence dye that easily crosses the cell membrane and reversibly accumulates into acidified membrane-bound compartments, such as lysosomes. Acridine orange gives fluorescence emission in a concentration-dependent manner, which is red at high concentrations (e.g., in lysosomes) to green at low concentrations (e.g., in the cytosol), with yellow as intermediate (e.g., upon trapping in nucleoli). The ratio of red-to-green emission in comparison with controls may thus either monitor lysosomal leakage or change in lysosomal pH. rPMs were grown in 96-well culture plates. rPMs were first exposed with acridine orange (5 μg/ml) at 37°C for 15 min, which were rinsed, then incubated in HBSS with or without cART and NAC for the indicated times. Cells were examined at 1 h intervals using a Synergy™ Mx Monochromator-Based Multi-Mode Microplate Reader (BioTek Instruments, Inc. Winooski, VT, USA) with excitation wavelength at 485 nm and emission recorded at 530 and 620 nm. To further confirm cART-mediated lysosomal membrane permeabilization (LMP), cells were stained with GAL3 and LAMP2.

Acridine orange is a fluorescent dye that easily traverses the cell membrane. It is a weak base, which reversibly accumulates into acidified membrane-bound compartments. The fluorescence emission of acridine orange is concentration-dependent, from red at high concentrations (e.g., in lysosomes) to green at low concentrations (e.g., in the cytosol), with yellow as intermediate (e.g., upon trapping in nucleoli). The shift in the red-to-green emission ratio in comparison to controls may thus either monitor lysosomal leakage or change in lysosomal pH. In our fluorometric studies, cells were cultured in 96-well culture plates. Cells were first exposed with acridine orange (5 μg/mL) at 37 °C for 15 min. Cells were rinsed, then incubated in HBSS with or without cART for the indicated times. Cells were examined at 1-h intervals using a Synergy™ Mx Monochromator-Based Multi-Mode Microplate Reader (BioTek Instruments, Inc. Winooski, VT, USA) with the excitation wavelength at 485 nm and emission recorded at 530 and 620 nm.

Growth of luminescent bacteria was measured as described previously [23 (link), 54 (link)]. Briefly, dilutions of 0.5–2.0 × 10⁵D. discoideum cells infected with M. marinum pMV306::lux were plated on a non-treated, white F96 MicroWell plate (Nunc) and covered with a gas permeable moisture barrier seal (Bioconcept). Luminescence was measured for 72 h at 1 h intervals with a Synergy Mx Monochromator-Based Multi-Mode Microplate Reader (Biotek). The temperature was kept constant at 25°C.