Glomax multi plus reader

The in vitro AChE inhibition assay was evaluated using a spectrophotometry method developed by Ellman with some modifications [41 (link)]. The assay was conducted in a 96-well plate with a total volume of 200 µL assay mixtures in comparison to galantamine as the positive control. Plant extracts were dissolved in dimethyl sulfoxide (DMSO) and tested at a final concentration of 0.2 mg/mL, while compounds were tested at 0.1 mg/mL. The final concentration of DMSO was fixed at 1% in the 96-well plate. In a 96-well plate, 178 µL of 50 mM phosphate buffer, 2 µL of 1 mg/mL compound and 20 mg/mL extract, and 10 µL of 0.5 U/mL AChE were added. The first incubation period was 15 min at 25 °C. Five microliters of 14 mM ATCI substrate and 10 mM of the color indicator, DTNB, were added equally into each well and the well was then incubated at 25 °C for 30 min to initiate the enzyme reaction. The absorbance was measured at 415 nm using Promega Glomax^® Multi Plus Reader (Promega, Madison, WI, USA). Each sample was done in triplicate and eight 2-fold serial dilutions were performed for each sample to determine the fifty-percentage inhibitory concentration (IC₅₀). The percentage of inhibition was calculated using formula (1) [42 (link)]: $\mathrm{Percentage}\mathrm{Inhibition}(\%)=\frac{Absorbance of negative control - Absorbance of test sample}{ Absorbance of negative control}\times 100$

The in vitro potential of acetylcholinesterase inhibitory activity was performed spectrophotometrically using Ellman’s method [90 (link),91 (link)]. The assay was conducted in a 96-well plate with a total assay mixture volume of 200 µL. Galantamine was used as the positive control. In a 96-well plate, an aliquot of 1 µL of extract (40 mg/mL DMSO) was mixed with 179 µL of 0.05 mM phosphate buffer, and 10 µL of 0.5 U/mL AChE (AChE from Electrophorus electricus (electrical eels), Type VI-S, 200–1000 unit/mg protein) was added to the designated wells. After 15 min incubation at 25 °C, 10 µL of equal amounts of 14 mM acetylthiocholine iodide (ATCI) substrate and 10 mM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) as a color indicator was added into each well and incubated at 25 °C for 30 mins to initiate an enzyme reaction. The absorption was measured at 415 nm using a Promega Glomax^® Multi Plus Reader (Promega, Sunnyvale, CA, USA). Each run was carried out in triplicate on three different days to determine the percentage of inhibition at 200 µg/mL. The % inhibition was determined using Equation (3): $\%\mathrm{Inhibition}=\frac{\mathrm{Abs}(-)\mathrm{ve}\mathrm{control}-\mathrm{Abs}\mathrm{test}\mathrm{sample}}{\mathrm{Abs}(-)\mathrm{ve}\mathrm{control}}\times 100$
Afterward, the IC₅₀ value for each sample showing AChE inhibitory activity of 50% or more was determined.

A previously set up luminometry-based method (Eklund et al., 2010 (link)) was used to compare the kinetic of mycobacterial growth harvested from either shaken or standing cultures during hMDMs infection. Aliquots of medium supernatant and cell lysate containing luciferase-expressing bacteria were transferred to pure water in white 96-well plates at selected time points. One percent of decanal (Sigma Aldrich) was injected through the instrument needle into each well and flash luminescence was measured using a Glomax Multiplus reader (Promega). Light emission, measured as arbitrary luminescence units (ALU), is linearly proportional to the number of viable bacilli. Both extracellular (supernatant) and intracellular (lysate) number of bacteria were obtained by subtracting the ALU values from uninfected cells. The ALU values from both supernatant and lysate were then standardized for dilutions and summed up to obtain total ALU values. To determine fold change of bacterial load, the median value of each triplicate of all time points was normalized to the initial time point of the same experiment.