Spectramax m2 multi mode microplate reader

MOLT4 cells (2 × 10⁶ cells/well) were seeded into sterile 6-well plate dishes for 24 hours before being treated with amygdalin bimetallic nanocomposites with IC₅₀ concentrations (30 and 40 μg/ml). Following the treatment protocol, MOLT4 cells were treated with 1 M of AO/EtBr at 37°C for 5 minutes in the dark. Finally, the stained cells were photographed and viewed using a fluorescence microscope's green and red channels (20X) (ZOE Cell Imager, BioRad, USA). The fluorescence intensity of AO/EtBr was measured in triplicate using a SpectraMax M2 Multimode Microplate Reader (Molecular Devices, USA).
Using rhodamine 123 dye, we investigated the permeability of mitochondrial membranes in nanocomposites-treated and untreated cells. The nanocomposites were applied to 6-well plates containing MOLT4 (2 × 10⁶ cells/well) cells and incubated for 24 hours at 37°C. After being rinsed twice in PBS, the cells were stained with 1 mM rhodamine 123 for 30 minutes before being incubated in the dark for 15 minutes. Images of fluorescent cells were produced after examining the dyed cells (20X) with a fluorescence microscope (ZOE Cell Imager, BioRad, USA). Three independent experiments were carried out to assess the intensity of Rh123 fluorescence using a SpectraMax M2 Multimode Microplate Reader (Molecular Devices, USA).

The ability of the peptides to prevent the initial bacterial attachment was done using an established protocol with minor modifications [44 (link),45 (link)]. In short, high-density overnight cultures of S. aureus MW2 were grown in TSB media. Forty microliters of this culture were added to each well of the microtiter plates containing 50 µl of serially diluted 2 × peptide solution. Media containing bacteria and water were treated as a positive control, while only media with water served as the negative control. The plates were then incubated at 37 °C for 1 h. The media were carefully pipetted out, and the wells were washed with PBS (GIBCO, MD, USA) to remove loosely attached planktonic cells, followed by the addition of XTT [2,3-bis(2-methyloxy-4-nitro-5-sulfophenyl)-2H-tertazolium-5-carboxanilide]. Quantitation of the inhibition of biofilm attachment was done by assay following manufacture instructions with minor adjustments (ATCC, VA, USA). The calorimetric intensity of the XTT dye at 450 nm was measured by the SpectraMax M2 Multi-mode Microplate Reader (Molecular Devices, CA, USA). The percentage of biofilm growth was plotted, assuming 100% biofilm growth is achieved in the bacterial wells without peptide treatment.