Zoe cell imager

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 morphological analysis of peloids was performed previously [12 (link)]. In the present study, Zoe Cell Imager (Bio-Rad) was used for peat samples (5 mg) resuspended in 0.5 mL of distiller water.
For the extraction process, 20 mL buffer (1.5 M Tris HCl, 0.4% SDS, 8 mM EDTA, pH 8.8) was added to 10 g of peat. Samples were mixed for 45 min at room temperature and then centrifuged for 10 min at 4000× g. The supernatants were collected for the analysis of total protein, thiol protein, and lipid content.
Total protein content was determined in accordance with the Lowry method using albumin as standard [13 (link)].
The concentration of thiol protein groups (protein-SH) was estimated using 5V-5V-dithio-bis(2-nitrobenzoic acid) (DTNB), as described by Di Monte et al. [14 (link)].
Nile Red was used for lipid detection and its fluorescence was measured using a Victor² Multilabel Counter Wallac (Perkin Elmer) [15 (link)]. Samples for lipid detection were prepared as follows: 5 μL sample and 5 μL Nile Red 1mg/mL were added to 190 μL of PBS buffer (sodium phosphate dibasic anhydrous 8 mM, sodium dihydrogen phosphate monohydrate 2 mM, 140 mM sodium chloride, pH 7.4). After incubation at room temperature (10 min), samples were loaded on a 96-well plate and read at 485 nm excitation and 572 nm emission.

Cells grown in monolayers under different conditions were fixed in 80% acetone (chilled at −20 °C) at 4 °C for 20 min. The cells were then washed three times with ice-cold PBS, blocked with 1% bovine serum albumin (BSA) for 30 min, and incubated with anti-SARS-CoV-2 Spike-RBD rabbit monoclonal antibody (diluted in PBS with 0.1% BSA at 1:1000; Sino Biological Inc, Beijing, China) at 4 °C overnight. The cells were again washed three times with ice-cold PBS and then stained with the Alexa Fluor488®-conjugated Goat Anti-rabbit IgG secondary antibody (diluted in PBS with 0.1% BSA at 1:1500; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature in the dark. The cells were washed three times and then incubated with 0.5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI, nuclear DNA staining; Solarbio Life Sciences, Beijing, China) for 5 min. The cells were washed three times; immunofluorescence pictures were captured using the Bio-Rad ZOE Cell Imager (Hercules, CA, USA) equipped with a fluorescence apparatus.