Uv 1750 spectrophotometer

Synechocystis sp. PCC 6803 and the knockout mutants constructed in this study were grown in BG11 medium (pH 7.5) under a light intensity of approximately 50 μmol photons m⁻² s⁻¹ in an illuminating incubator of 130 rpm at 30°C (HNY-211B Illuminating Shaker, Honour, China) (Qiao et al., 2012 (link)). Cell density was measured on a UV-1750 spectrophotometer (Shimadzu, Japan) at OD₇₃₀ or on an ELx808 Absorbance Microplate Reader (BioTek, Winooski, VT, USA) at OD₆₃₀. For growth and ethanol treatment, 40 μL fresh cells at OD₆₃₀ of 0.2 collected by centrifugation and were then inoculated into 200 μL of BG11 liquid medium in 96-well cultivation plates. Ethanol at a final concentration was added at the beginning of cultivation. The 96-well cultivation plates were fixed in the shaker and measured directly on ELx808 Absorbance Microplate Reader at OD₆₃₀ every 12 h. To ensure accuracy of finding, the mutants with differential growth patterns under ethanol stress were then confirmed by growth in 250-mL flasks, in which 10 mL fresh cells collected by centrifugation and were inoculated into 50 mL of BG11 liquid medium in a 250 mL flask. Culture samples (1 mL) were taken and measured at both OD₇₃₀ and OD₆₃₀ every 12 h. Growth experiments were repeated at least three times to confirm the growth patterns.

The anthocyanin concentration was measured using a modified version of the pH-differential procedure [58 (link),59 (link)]. The freeze-dried tissue (approximately 1.0 g) was extracted at 4 °C for 12 h using 5 mL of extraction solution (0.3% HCl/methanol). Following 15 min of centrifugation at 10,000× g, the resulting product was shifted to a sterile tube and the particulates extracted two or three times with extraction solution until no red color remained in the precipitate. The combined supernatants were diluted to 25 mL. A UV-1750 spectrophotometer (Shimadzu, Japan) was used to measure absorbance at 510 nm and 700 nm in pH 1.0 and pH 4.5 buffers. The anthocyanin concentration was determined in terms of cyanidin-3-O-glucose equivalent using the following equation: TA (mg/100 g) = A × MW × 5 × 100 × 25/e, where TA represents the total quantity of anthocyanin and A = [(A₅₁₀ − A₇₀₀)_pH1.0 − (A₅₁₀ − A₇₀₀)_pH4.5]. The molecular weight (MW) was 449.2 and the molar absorption (e) was 26,900. Each biological replicate sample was measured three times.

All chemical reagents in the syntheses were purchased from Energy Chemical (Shanghai, China) Co., Ltd., and used without purification unless otherwise. Solvents were dried by standard methods prior to use. MitoTracker Green FM and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were all obtained from Thermo-Fisher Biochemical Products (Beijing, China) Co., Ltd. ¹H NMR and ¹³C NMR spectra of synthetic intermediates and final probe were recorded on Agilent DD2 (Agilent Technologies, Santa Clara, CA, USA) (600 MHz, 150 MHz). Electrospray ionization mass spectrometry (ESI-MS) was operated on a Bruker maXis 4G mass spectrometer (Bruker Daltonik Gmbh, Bremen, Germany). pH measurements were made on a Sartorius PB-10 pH meter (Göttingen, Germany). UV-Vis absorption spectra were recorded on a SHIMADZU UV-1750 spectrophotometer (Kyoto, Japan). Fluorescence spectra were taken on a PerkinElmer LS-55 spectrofluorometric (Waltham, MA, USA). HeLa cells were cultured in a Thermo Forma, model 371 CO₂ incubator (Thermo Fisher Scientific, Waltham, MA, USA). The absorbance for an MTT assay was obtained using a Thermo Scientific Varioska Flash microplate reader (Waltham, MA, USA). Confocal microscopy fluorescence images and two-photon fluorescence images were taken using an Olympus FV1000-MPE multiphoton laser scanning confocal microscope (Olympus Co., Ltd., Nagano, Japan).