2.6. Live/dead assay

Calcein‐AM and propidium iodide (PI) were used to identify live and dead cells, respectively (Cellstain Double Staining Kit; Dojindo Laboratories). An assay solution consisting of calcein‐AM and PI was prepared by adding 10 μL of 1 mmol/mL calcein‐AM stock solution and 15 μL of 1.5 mmol/mL PI solution to 5 mL of HBSS(+). NHEK‐Ad cells were pre‐cultured for 24 hours and washed twice with HBSS(+) before treatment with 1 mL/well of 0.5 mM C8K, C18:1K, SLES or SLS for 5 minutes at room temperature. The cells were washed three times with HBSS(+) and stained with 0.5 mL of the assay solution for 15 minutes. ¹⁸ The cells were washed with HBSS(+) three times and fresh HBSS(+) was added to each well. The double‐stained cells were assessed by fluorescence microscopy (Biorevo BZ‐9000, Keyence Corporation) (Figure 3).

Live/dead assay procedure for NHEK‐Ad cells. NHEK‐Ad cells pre‐cultured in KGM‐Gold medium for 24 hours were treated with 1 mL/well of each sample for 5 minutes. NHEK‐Ad cells were washed three times with HBSS(+), and stained with calcein AM and propidium iodide for 15 minutes. After staining, the cells were washed three times with HBSS(+) and photographed using a fluorescence microscope