Tma dph

Benzaldehyde (BEN > 99%) was purchased from Chroma Biotechnology Co., Md (Chengdu, China). Parahydroxybenzoic acid (>98%), benzaldehyde (BEN > 99%), acyclovir (ACV > 99%), carbamazepine (CBZ > 99%), vinblastine (VIN > 99%), hydrochlorothiazide (HTZ >99%), and propranolol hydrochloride (PRO >99%) were purchased from Chengdu Alfa Biotechnology Co., Ltd. (Chengdu, China). TMA-DPH was purchased from MedChemExpress (MA, United States). Acetonitrile and formic acid (HPLC grade) were bought from Fisher Chemical (MA, United States). HBSS and MEM were bought from Hyclone (MA, United States), nonessential amino acids solution was purchased from Sigma-Aldrich (Shanghai, China), and sodium pyruvate was bought from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). FBS was purchased from Every Green Biotechnology Co., Ltd. (Zhejiang, China). 0.25% Trypsin–EDTA and penicillin–streptomycin solution was provided by Gibco (NY, United States). DMSO was obtained from Meilunbio (DaLian, China). A 12-well Transwell^® plate was purchased from Corning (NY, United States). All other solvents were of analytical agents and aqueous solutions were prepared by double-distilled water.

Yeast cells were grown at 42 °C in 100-ml Erlenmeyer flasks containing 50 ml YP medium with 200 g/l glucose at 220 rpm. For measuring trehalose accumulation, cells were harvested at stationary phase after incubation for 36 h, when cells accumulate high levels of trehalose as previously reported [60 ]. Trehalose levels were determined using trehalose content detection kit (BestBio, China) in accordance with the manufacturer’s instructions. For determining membrane fluidity, cells were harvested after incubation for 8 h, 16 h and 36 h at the early-exponential, mid-exponential and stationary phases, respectively. Membrane fluidity was assessed using steady-state fluorescence spectroscopy. Steady-state anisotropy of 1-[4-(trimethylamino)pheny]-6-phenyl-1,3,5-hexatriene (TMA-DPH, MedChemExpress, USA) following incorporation of the probe into yeast plasma membranes was measured, as previously described with a slight modification [61 (link)]. A Spark™ Multimode Microplate Reader (Spark 10 M, Tecan, Switzerland) was used for the measurement of the steady-state anisotropy of TMA-DPH. Both labelling of cells with TMA-DPH and the measurement were conducted at 42 °C.

Mitochondria were first isolated using mitochondria isolation kit (Beyotime, Shanghai, China). Mitochondria membrane fluidity was measured by monitoring the fluorescence polarization (P) of trimethylammonium 1,6-dipheny-1,3,5-hexatriene dye (TMA-DPH, Medchem Express). The mitochondria were treated with 5 μM TMA-DPH. Fluorescence polarization value was measured using FluoroMax®-Spex 3 spectrofluorometer (Jovin Yvon Horiba, Edison, NJ, USA), at an Ex/Em wavelength of 355/430. P was calculated from the fluorescence intensity (FI) according to the following formula: $$P=\left(Ivv-G\ast Ivh\right)/\left(Ivv+2G\ast Ivh\right)$$
Ivv and Ivh represented the excitation and emission fluorescence intensity polarization in vertical (v) and horizontal (h), respectively. G is the apparatus constant dependent on the emission wavelength. P value and membrane fluidity are negative correlation.