Tma dph

The plasma membrane fluidity of HeLa cells was estimated by means of the fluorescence anisotropy of the hydrophobic probe TMA-DPH (1-4-trimethylammoniumphenil-6-phenil-1,3,5-hexatriene; Thermo Fisher Scientific). HeLa cells (70–80% confluence) were incubated with L. crispatus BC5, S. agalactiae, E. faecalis, or B. subtilis cells (5 × 10⁷ CFU) for 1 h at 37°C with 5% CO₂, then washed three times with PBS and re-suspended at a final concentration of 3 × 10⁵ cells/mL. The absorbance of the cell suspension was kept lower than 0.15 at the excitation wavelength of TMA-DPH. A few microliters of TMA-DPH stock solution were added to the cell suspension in order to obtain a final probe concentration of 1 μM. Fluorescence anisotropy measurements were performed by using a PTI QuantaMaster fluorimeter (Photon Technology International, North Edison, NJ, United States) equipped with a temperature-controlled cell holder and Polaroid HNP’B polarizers. Temperature was kept at 25°C. Excitation and emission wavelengths were set at 360 and 430 nm, respectively.

Steady-state fluorescence anisotropy was used to investigate the possible modifications induced by PBS or sugar solutions on the physico-chemical state of CT EB membranes. The membrane fluidity of HeLa cells and CT EBs was estimated by measuring fluorescence anisotropy of the hydrophobic probe TMA-DPH [1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate] (ThermoFisher Scientific, Waltham, MA). TMA-DPH is a lipophilic fluorophore that penetrates the membrane hydrophobic core, orienting perpendicularly to the membrane plane [36 (link)]. In case of an increased fluidity of the membrane, the TMA-DPH probe rotates to a greater extent, leading to a depolarization of the fluorescence emission and a decrease in the fluorescence anisotropy.
Anisotropy was measured after 2 h-incubation with sugar solutions (glucose, sucrose, and mannitol 5 mM) for 2 h, at 37 °C, with 5% CO₂. A PBS solution without sugars was used as a control.
HeLa cells and EBs were suspended at a final concentration of 3 × 10⁵ cells/mL and 5 × 10⁴ cells/mL, respectively. For the anisotropy measurements, the samples were incubated with TMA-DPH and then analyzed by a PTI QuantaMaster fluorimeter (Photon Technology International, North Edison, NJ, USA) according to Parolin et al. [30 (link)].