At least 10,000 cells were analyzed for each Mab staining using a FACScan flow cytometer (Becton Dickinson,

Franklin Lakes, NJ, USA). Detection of cytokines Thymocyte suspension was prepared from thymocytes in the RPMI-1640 medium. The suspension of thymocyte and splenocyte was adjusted to 1 × 107 and 2 × 107 cells/ml, respectively, and planted into the 24-well flat-bottom plate (0.5 ml per well). selleck screening library ConA was added to the final concentration of 5 μg/ml to introduce cytokine secretion. The cells were cultured for 48 h at 37°C in a humidified incubator containing 5% CO2 at 37°C. The supernatant of each well was collected for cytokine analysis. IL-4 and IFN-γ ELISA kits were used. Briefly, 50-μl samples or standard control were mixed with 50-μl assay diluents

https://www.selleckchem.com/products/Y-27632.html and incubated at 37°C for 90 min. After being washed five times, 100-μl antibody-labeled biotin was added to each well. The plate was incubated for 60 min. Following five times of rinsing, a 100-μl substrate solution was added to each well and incubated for 30 min. Finally, a 100-μl stop solution was added to each well, and the colored reaction product was measured at 450 nm on a microplate reader (Thermo Fisher Scientific Inc.). The expression level of cytokine analysis by Western blot Fresh spleens of mouse in each group were stored in ice-cold tubes, and then the total proteins were extracted from the organs. The protein concentration was analyzed using BCA protein assay kit. The proteins in the spleen extracts were separated by 10% SDS-PAGE and electrophoretically transferred Aspartate onto a polyvinlidene difluoride membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% nonfat milk in TBS containing 0.1% Tween 20 at 37°C for 2 h followed by incubation

overnight at 4°C with antibodies against IL-12, IFN-γ, IL-4, and TNF-α. β-Actin was taken as the reference protein. The membranes were washed with TBS containing 0.1% Tween 20 and probed with horseradish peroxidase-labeled goat anti-rabbit or anti-mouseIgG. The proteins were detected with enhanced chemiluminescence imaging. Statistical analysis Data were analyzed using the Statistical Package for Social Science (version 19.0; SPSS Inc., IBM, Armonk, NY, USA). The significant difference between groups was analyzed using one-way ANOVA; P < 0.05 was considered statistically significant. Results and discussion Results The characteristic of carbon dots As shown in Figure 1, the UV–vis absorption spectra of carbon dots and photoluminescence (PL) emission spectra excited by various incident lights are shown in Figure 1a,b, respectively. At an excitation wavelength of 340 nm, a strong emission peak at about 430 nm was observed in the PL emission spectrum of carbon dots.