Dm575

The GFP and DsRed immobilized on the Ni-MPA-gold substrates were observed under a fluorescence microscope (Nikon ECLIPSE TE2000-U) equipped with 4 × 0.13 numerical aperture (NA) objective lenses. The immobilized His-tagged GFP (Excitation: 475 nm; Emission: 509 nm) was observed with a B-2A excitation wavelength (EX470/40, DM505, and EM520; Nikon) and the immobilized His-tagged DsRed (Excitation: 556 nm; Emission: 586 nm) was observed with a G-2A excitation wavelength (EX535/50, DM575, and EM590; Nikon). Fluorescent images were captured with a digital camera (Nicon D80), and the amount of immobilized His-tagged GFP and DsRed was estimated by analyzing the fluorescence intensity using Image J free software (NIH; http://rsbweb.nih.gov/ij/). The calibration line was obtained by polynomial fitting of the fluorescence intensities of standard samples. The scale was determined by comparison with the appropriate scale on the captured images. After the observation, the substrates were soaked in 0.5 M imidazole solution to dissociate the immobilized His-tagged GFP and DsRed and incubated at 37 °C overnight with stirring while shielded from light. The imidazole-treated substrates were observed under the fluorescence microscope and the images were captured with the digital camera.

After chemical exposure, the epidermis of the RHS‐LCs was separated from the hydrogel using forceps. Subsequently, the epidermal sheet was submerged in fluorescence‐activated cell sorting (FACS) buffer (PBS supplemented with 0.1% bovine serum albumin and 0.1% sodium azide) for 1 hour at 4°C (maximum overnight) with 100 μL/mL phycoerythrin (PE)‐labelled antigen CD1a (BD Pharmingen, San Diego, California) in FACS buffer and examined with a fluorescence microscope (Nikon Eclipse 80i, G‐2a Ex510‐560, DM575, BA590). The density of MUTZ‐LCs was determined by calculating CFSE⁺/CD1a⁺ cells based on their fluorescence intensity with NIS‐Elements software (Nikon Instruments Europe, Amsterdam, The Netherlands).