## 1 Introduction
#### 2.1.8 In Vitro Cytotoxicity/MTT Assays  
The cytotoxic effects of the AgNPs were assessed in cervical carcinoma cells (HeLa), human embryonic kidney (HEK293) cells, and breast adenocarcinoma (MCF-7) cells. Cell lines (Cryopreserved) were obtained from the American Type Culture Collection (ATCC) situated in Manassas, VA, USA. The experimental procedures involving cell cultures were conducted within a sterile class II biohazard safety cabinet. Cryopreserved cell lines, which were maintained at \(-\)80 \({}^{\circ}\)C in a Nuaire biofreezer, were retrieved and then defrosted at 37 \({}^{\circ}\)C in a water bath. Aseptically, the cell suspensions were placed into falcon centrifuge tubes and centrifuged (Eppendorf benchtop centrifuge) for 5 min at 1000 rpm, after which the supernatant was discarded. The resulting pellet was then re-suspended in 1 mL of whole sterile medium (Eagle's Minimum Essential Medium (EMEMEM)) augmented with 1% antibiotics (100 \(\upmu\)g/mL streptomycin, 100 units/mL penicillin,) and 10% Foetal Bovine Serum (FBS). The cell suspensions were subsequently dispensed and cultured in a 25 cm\({}^{2}\) tissue culture flask which contained 4 mL of whole sterile medium. These cells were then incubated at 37 \({}^{\circ}\)C (Thermo-Electron Corporation, Waltham, MA, USA) in an atmosphere containing 5% CO\({}_{2}\) and observed daily using an inverted microscope (Nikon TMS-F 6V, Tokyo, Japan). The sterile whole medium was routinely replaced until the cells reached confluency [65].  
MTT (cell viability) assay protocol  
The assay employed aimed to quantify the metabolic activity of cells and their ability to reduce MTT into formazan using the succinate-tetrazolium reductase system [66]. The cells were subjected to trypsinization and subsequently seeded into 96-well microtiter plates, where they were incubated overnight at 37 \({}^{\circ}\)C to allow for cell attachment. Thereafter, the medium was substituted with new medium (EMEM + 10% FBS + 1% Antibiotics) [65]. Cells were then treated with different concentrations of synthesized AgNPs from _B. albostellata_ (15, 30, 60, 120, and 140 \(\upmu\)g/mL) and incubated for 48 h at 37 \({}^{\circ}\)C. In each well, the growth medium was aspirated and 100 \(\upmu\)L of medium (comprising 10 \(\upmu\)L of the MTT solution (5 mg/mL in phosphate buffered saline (PBS) solution) was added, followed by a 4 h incubation at 37 \({}^{\circ}\)C. Subsequently, the medium containing MTT was removed from each well and replaced with 100 \(\mu\)L of DMSO to ensure a complete solubilization of the formazan crystals. The formation of these crystals served as an indicator of cellular viability [67]. Once DMSO was added, the solution exhibited a purple coloration. The absorbance of the solution was measured at 570 nm using the Mindray M-R-96A microplate reader (Vacutec, Hamburg, Germany) and DMSO as the control. Solutions which contained cells only served as the positive controls and was recorded as 100% survival [65]. This protocol was conducted in triplicate. Graphs were created using Microsoft Excel 2019(tm) in order to determine the concentration at which 50% cell death was attained (IC50).