F 2700 fluorescence spectrophotometer

In a typical procedure [29 (link)], 25 μL of 0.1 M AgNO₃ was added to 10 mL 0.625% (wt) PEI stock solution and the solution was continually stirred for 2 h to ensure thorough complexation between Ag⁺ and the amine ligands. Then, the solution pH was adjusted to 5 with HAc solution. After 30 μL of 0.1 M AA was added to the solution, the continuous stirring was kept for 2 days. Altering the PEI molecular weights from 0.6 kDa to 25 kDa under same mass concentration conditions would produce PEI0.6k-AgNCs, PEI1.8k-AgNCs, PEI10k-AgNCs and PEI25k-AgNCs, respectively. AgNPs were also prepared according to the previous literature with slight modifications [33 ]. Briefly, 25 μL of 0.1 M AgNO₃ was dripped to 10 mL solution containing 0.008 M NaBH₄ while stirring in an ice bath at approximately one drop per second. After AgNO₃ is added, 100 μL of 6% PVP was injected into solution to stabilize the AgNPs. These two nanomaterials were characterized systematically. The UV–Visible absorption spectra of PEI-AgNCs and AgNPs were obtained with a UV-1800 spectrophotometer (Suzhou Shimadzu Instrument Co., Ltd., Suzhou, China). The fluorescence spectra were measured using a F-2700 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). High resolution transmission electron microscopy (HRTEM) images were collected with a H-7500 high resolution transmission electron microscope (Hitachi).

The intrinsic fluorescence spectroscopy of TPSPH and five fractions was measured using the method of Liu et al. [16 (link)] with a fluorescence spectrophotometer (F2700 fluorescence spectrophotometer, Hitachi Co. Ltd., Tokyo, Japan). The samples (0.03 mg/mL) prepared in a 0.01 M phosphate buffer (pH 7.0) were scanned in the wavelength range of 300–400 nm with an excitation wavelength of 280 nm and a slit width of 10 nm.

RBL-MRGPRX2 cells (2 × 10⁶ cells) were loaded with Fura-2 acetoxymethyl ester (1 μM) in HEPES buffer containing 0.1% BSA for 30 min in the dark at 37°C, followed by 15 min period for de-esterification in dark at room temperature. Cells were washed and resuspended in HEPES-buffered saline and Ca²⁺ mobilization was measured for the designated time. Calcium mobilization was determined by measuring the fluorescence ratio between dual excitation wavelengths of 340 and 380 nm, and an emission wavelength of 510 nm using a Hitachi F-2700 Fluorescence Spectrophotometer (29 (link)).

Extraction and purification of Flamindo/Flamindo2 protein and in vitro spectroscopy were performed as previously reported [20] (link). Briefly, a Flamindo/Flamindo2-containing pRSET_B vector was introduced into Escherichia coli JM109 (DE3) cells and cells were cultured at 20°C for 4 days. After 4 days of culture, cells were harvested by centrifugation, lysed by three freeze-thaw cycles, and sonicated with lysozyme. After centrifugation of the lysate, His-tagged Flamindo/Flamindo2 protein was purified from supernatants using a Ni-NTA agarose column (Qiagen) followed by a PD-10 gel-filtration column (GE Healthcare). The purified protein was finally eluted in Hepes buffer (150 mM KCl and 50 mM Hepes-KOH [pH 7.4]). The concentration of purified Flamindo/Flamindo2 protein was measured by the Bradford protein assay using Bio-Rad Protein Assay (Bio-Rad). Bovine serum albumin was used as a standard. Absorption and fluorescence spectra of purified Flamindo/Flamindo2 protein were measured using a UV-670 UV-Vis spectrophotometer (Jasco) and F-2700 fluorescence spectrophotometer (Hitachi), respectively.

Fluorescence measurements were conducted at room temperature on an F-2700 fluorescence spectrophotometer (Hitachi, Japan). The fluorescence emission spectra were recorded in the range of 400–570 nm with the excitation wavelength set at 370 nm. The slit widths for excitation and emission were both set at 10 nm.