Tcs sp8 x confocal spectral microscope imaging system

Cells grew on slides were fixed using 4% paraformaldehyde in PBS for 10 min at room temperature. Permeabilization of cell membrane was created by incubating cells with 0.25% Triton X-100 in PBS for 5 min. The blocking reaction was performed using 1% BSA-containing PBST for 30 min, and then cells were incubated with primary and secondary antibodies. Mounting medium contains DAPI for nuclear DNA staining. The fluorescence signals were analyzed using Leica TCS SP8 X confocal spectral microscope imaging system.

The fluorescence
and UV–vis spectra of the PDs were recorded using an F-2700
fluorescence spectrophotometer and a Lambda 265 UV–vis spectrophotometer
(PerkinElmer), respectively. The particle sizes of the PDs were measured
using a dynamic light scattering (DLS) instrument (Zetasizer Nano
ZS90, Malvern). Transmission electron microscopy (TEM) was performed
using a JEM-1400 microscope (Japan) operated at an acceleration voltage
of 120 kV. Zeta potentials were recorded using a nanoPartica SZ-100V2
instrument (HORIBA). The structures of the PDs were identified using ¹H NMR spectroscopy (Agilent Technologies, 600 MHz), FTIR spectroscopy
(IRAffinity-1S, Shimadzu), and XPS (PHI 5000 VersaProbe, ULVAC-PHI).
Surface tensions were measured using a DST-30 digital surface tension
analyzer (Surface Electro Optics). Confocal microscopy images were
recorded using a Leica TCS SP8 X confocal spectral microscope imaging
system and a white-light laser.
The QYs of the PDs were determined
from the integrated fluorescence intensity and the absorption, according
to the relative fluorescence QY equation where Φ_PD is the QY of the
PD, Φ_st is the QY of quinine sulfate in 0.1 M H₂SO₄ (Φ_st = 0.54), I is the slope of the plot of the integrated fluorescence intensity
with respect to the absorbance, and η is the refractive index
of the solvent.

For pHyPer-cyto vector detection, tumor cells (2 × 10⁵ cells) were transfected with 2.5 μg pHyPer-cyto plasmid (Cat.No. FP941, Evrogen) with Lipofectamine 2000 reagent (Thermo Fisher) for 6 h, and then transferred to normal culture medium for 24 h. After PBS wash and Hoechst 33342 staining, the fluorescence generated by the binding of pHyPer-cyto vector and H₂O₂ was detected using Leica TCS SP8 X confocal spectral microscope imaging system.
For flow cytometer detection, trypsinized tumor cells (3 × 10⁵ cells) were treated with 10 μM CM-H2DCFDA for 30 min, and then analyzed using BD FACSCalibur flow cytometer system and CellQuest software.