2.8.3. Membrane Fluidity by TMA-DPH Fluorescence Anisotropy Assay

We used the steady-state fluorescence polarization method [34]. In brief, HaCaT cells were cultivated in 6-well plates (1 × 10⁶ cells/well) and pretreated with NAR for 24 hr. Cells were gathered 2 min after UVB irradiation by gentle scraping in PBS with a cell lifter which left them undamaged. Cells were then labelled with the fluorescent probe TMA-DPH (2 μM). Membrane fluidity was assessed 1 min after cell labelling on a fluorescent spectrophotometer equipped with polarizers (Perkin-Elmer, France). Samples were excited by exposure to vertically polarized light (360 nm), and emitted light was analyzed at 435 nm vertically and horizontally to the direction of the excitation. All fluorescent measurements were carried out at 23°C.

Anisotropy values (r) were calculated as follows:

where I_VV is the emission intensity that is vertically measured, I_VH is the emission intensity that is horizontally measured, and G is the correction factor that is used for calculations in an optical system.