Carboxyfluorescein succinimidyl ester (cfse)

The trophozoites were incubated with 5 μM CFSE (ThermoFisher Scientific) in PBS with 1% BSA on a gentle rotation at 37°C for 30 min. After washing three times with PBS to remove the excess dye, the parasites were suspended in keratinocyte serum-free medium (Gibco) for analysis. The CFSE-labeled parasites were co-cultured with hVECs at the multiplicity of infection (MOI) of 2 in a minimal thin layer of keratinocyte serum-free medium for 1 hr in 5% CO₂. After washing the unbound trophozoites away with pre-warmed PBS, the CFSE signal was detected by inverted fluorescence microscopy (CFSE Ex/Em = 492/517) (Axiovert 200M, Zeiss). For each assay, the signal intensities quantified by ImageJ v.1.53k software (National Institutes of Health) in five independent microscopic fields were averaged to evaluate the relative parasite cytoadherence. The signal from the control parasites was defined at 100%.

INS-1 832/13 cells were stained with a fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) (#C34570, Thermofisher Scientific). Due to its covalent coupling reaction to intracellular molecules, fluorescent CFSE is retained within cells once incorporated. Briefly, the cells were incubated with 5 µM CFSE in Krebs buffer containing (mM) 120 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 KH₂PO₄, 1.2 MgSO₄, 2.8 glucose, 25 NaHCO₃, 10 HEPES (pH 7.4 with NaOH) for 30 min at 37 °C with 5% CO₂. The cells were washed twice with Krebs buffer (as above) with 1 mg/ml BSA. Next, the cells were incubated for a further 10 min to allow CFSE to undergo full hydrolysis. Subsequently, the cells were perfused at a rate of 1 ml/min at 32 °C with Krebs buffer containing 2.8 or 16.7 mM glucose, respectively. The fluorescence from CFSE was monitored every min at excitation wavelength 492 nm and the emitted light signals were read at 517 nm long-pass filter on confocal microscopy (LSM 510, Carl Zeiss, Germany). The cell area was measured by ImageJ software (version 1.52a).

For the in vivo competition assay, CD3⁺ T cells were isolated from three littermate piglets and cocultured with DCs (treated or not with PEDV). Then, the CD3⁺ T cells were purified and labeled with CFSE (green) or CM-DiI (red, Molecular Probes), respectively, for long-term cell labeling. Equal numbers of cells (1 × 10⁷) from the two populations were then mixed and adoptively transferred into piglets (autologous) via front cavity vein injection. The recipient piglets were sacrificed 24 h after the injection. For the flow cytometry analyses, cells were isolated from PBMCs, duodenum, jejunum, and ileum. For histological analyses, the duodenum, jejunum, and ileum were washed with 0.1 M PBS (pH 7.4) and embedded in OCT compound (Sakura Finetechnical). The frozen tissues were cut into 10-μm sections and mounted onto poly-L-lysine-coated glass slides. The CM-DiI or CFSE-labeled cells were observed with a fluorescence microscope (Carl Zeiss). Investigators were not blinded to experimental groups. Pathologists interpreting slides were blinded to PEDV-treated and control animal identifiers.