2.5.6. Cytotoxicity Test of Leather/SeNPs

Cytotoxicity was evaluated on human normal melanocyte cell line (HFB4) using MTT assay [45]. The leather/SeNPs dyed under the optimum conditions were sterilized and cut. Then it was plated on the six-well tissue culture plate. This plate was inoculated with cells and incubated for 24 h at 37 ◦C. The cell monolayer was washed twice using washing media after growth medium decantation. The cytotoxicity physical signs were checked in the tested cells. The tissue was picked up, and then 20 μL of 3-(4,5-dimethylthiazol-2 yl)-2,5-diphenyltetrazolium bromide dye (MTT) dissolved in phosphate buffer saline at a concentration of 5 mg/mL was added to each well with shaking for 5 min and incubated at 37 ◦C and 5% CO2 for 5 h. The formazan was formed as MTT metabolite and resuspended in 200 μL dimethyl sulfoxide with shaking for 5 min. The absorbance was measured at 560 nm and the background was read at 620 nm, and then subtracted.

#### 2.5.7. Antibacterial Activity

The antibacterial activity test for leather/SeNPs was conducted according to AATCC (147-2004) [46]. The bacterial strains were spread on the media using a sterile cotton swab. Antibacterial activity was evaluated against G+ve bacteria (*Bacillus cereus*) and G-ve bacteria (*Escherichia coli*, *Salmonella typhi and Pseudomonas aeruginosa*). Leather/SeNPs samples were cut to be square in shape and measuring 10 × 10 mm, while standard drugs such as tetracycline and ciprofloxacin were loaded in the disks to compare between their antibacterial activity and that of the tested leather/SeNPs. The petri dishes were then incubated at 37 ◦C for 24 h. while the growth inhibition zone diameters (mm) were determined.
