Neutral red uptake assay

In order to evaluate the cytotoxicity of the tested hydrogels on HaCaT and BJ cells, the Neutral Red Uptake Assay (Sigma Aldrich, Poznan, Poland) was used. This test was performed based on the procedure described previously [67 (link)]. After 2 and 24 h of exposure to extracts of the six GEN-loaded hydrogels, cells were incubated for 2 h with a neutral red dye (40 µg/mL), which was dissolved in serum-free DMEM medium. After incubation with NR, cells were washed with sterile PBS and 150 µL of decolorizing buffer (C₂H₅OH/CH₃COOH/H₂O, 50%/1%/49%) was added to each well to release cellular dye into the PBS solution. After shaking the cells for 15 min the absorbance of the dissolved dye at λ = 540 nm was determined using a FilterMax F5 Multi-Mode microplate reader (Thermo Fisher). The mean absorbance of the control cells (untreated with the extracts) was taken as 100% cell viability and used to calculate the percentage of viable cells in the experimental samples treated with the test hydrogels. As a part of the study, three independent experiments were carried out, in which each concentration of extracts was tested using four replicates.

The neutral red uptake assay (Sigma Aldrich) was used to assess HaCaT and fibroblasts viability. This assay is based on the initial protocol described by Borenfreund and Puerner.^{28 (link)} It allows to evaluate cell viability and determine accumulation of the neutral red dye in the lysosomes of viable, uninjured cells. The examined cells were placed in 96-well plates at a density of 1 × 10⁴ cells/well. After 24 h of preculture, medium (DMEM or MEM) was aspirated and tested concentrations of M. sativa L. UAE seeds and herb extracts (in the range of concentrations from 0.25% to 10%) were added into each well and cultured for another 24 h. The control group was unexposed cells. Following exposure to tested extracts, cells were incubated for 2 h with neutral red dye (40 μg/mL) dissolved in serum-free DMEM or MEM medium. Then, cells were washed with PBS and 150 μL of destain solution (EtOH/AcCOOH/H₂O₂, 50%/1%/49%) per well was added. The plates were shaken gently for 10 min until the neutral red had been extracted from the cells. Neutral red dye uptake was determined by measuring the optical density of the eluted dye at λ = 540 nm in microtiter plate reader spectrophotometer FilterMax F5 (Thermo Fisher). The experiments were performed in triplicates for each extract concentration and presented as percentage of control values (100%).

The second test used to evaluate the cytotoxicity of the analyzed extracts was the Neutral Red Uptake Assay (Sigma Aldrich). The research was carried out according to the procedure previously described by Repetto et al. [76 (link)]. After plating both cell types in 96-well plates (10⁴ cells per well) and 24-h incubation, the DMEM medium was removed and replaced with PRE, PGE, KTE, CTE and GGE extracts dissolved in DMEM medium. The analyses used extracts at concentrations of 50, 250 and 500 μg/mL. After 24 h of exposure of the cells to the test extracts, they were aspirated and replaced with neutral red dye (40 µg/mL) dissolved in serum-free medium (DMEM) and incubated for 2 h. The cells were then washed twice with phosphate buffered saline (PBS), then 150 μL of decolorizing buffer (EtOH/AcCOOH/H₂O, 50% (v/v)/1% (v/v)/49% (v/v) was added to each well to extract neutral red retained inside fibroblast and keratinocyte cells. The uptake of neutral red dye was determined by measuring the optical density (OD) at λ = 540 nm in a FilterMax F5 microtiter plate spectrophotometer (Thermo Fisher). The analyses were performed in three independent experiments in which each sample was examined in four replications. The results are presented as a percentage of the control value which is the optical density of cells untreated with the extracts (100%).