Carboxyfluorescein diacetate succinimidyl ester cfse dye

A carboxyfluorescein diacetate succinimidyl ester (CFSE) dye (Thermo Fisher Scientific) was used as a fluorescence tracking approach to detect the EpSC proliferation [29 (link)]. Briefly, EpSCs were labeled with a 5 μM CFSE dye solution for 20 min at 37 °C in the dark. Then, the cells were washed with RPMI medium containing 10% FBS to remove residual dye. The labeled EpSCs were treated for 3 days with DETCs (EpSCs:DETCs = 10:1) or DETCs-derived exosomes (0, 5, 15, 45 μg/mL). To determine cell proliferation, EpSCs were harvested and detected by flow cytometry. The expression levels of CFSE were analyzed by calculating the mean fluorescence intensity (MFI) values using a FlowJo software. The greater MFI values indicated the lower proliferation rate.

Model antigen OVA was purchased from Sigma-Aldrich (St. Louis, MO, USA), CpG 5′-TCCATGACGTTCCTGACGTT-3′). FAM-labeled CpG and control ODN (5′-TCCATGAGCTTCCTGAGCTT-3′) were obtained from Sangon (Shanghai, China). Carboxyfluorescein diacetate succinimidyl ester (CFSE) dye and the Micro BCA protein assay kit (BCA) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fluorochrome-conjugated anti-mouse antibodies were obtained from eBioscience (San Diego, CA, USA) and BioLegend (San Diego, CA, USA). Recombinant mouse GM-CSF and IL-4 were obtained from PeproTech (Rocky Hill, NJ, USA). Cy5-SE was purchased from Fanbo Biochemicals Company (Beijing, China). GSH, ethanol, and NaCl were all of analytical grade. The medium for culturing dendritic cells, splenocyte cells, and tumor cells was RPMI 1640/DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 U/mL penicillin (Invitrogen), and 100 μg/mL streptomycin (Invitrogen).