Fitc labeled anti cd11c

DC phenotypes were confirmed by flow cytometry. Cell suspensions were washed with PBS after 48 h of co-culture, and the cells were resuspended in PBS at a density of 10⁶ cells/ml. CD11c and CD83 are surface antigens of DCs and mature DCs^{19 (link), 20 (link)}. Fluorescein isothiocyanate (FITC)-labeled anti-CD11c and PE-labeled anti-CD83 (BioLegend Inc., San Diego, USA) solutions were added to the suspensions according to the manufacturer’s instructions. Following incubation for 20 min in complete darkness at room temperature, 2 ml of PBS was added, and the tubes were centrifuged for 5 min at 158 × g. The cells were immediately analyzed using a FACScan flow cytometer (BD FACS Aria, BD Biosciences, CA, USA) following resuspension in PBS (Fig. 3).Figure 3

Flow cytometry detection of the phenotypes of DCs and T lymphocytes before co-culture.

Flow cytometry was used to detect the expression of major histocompatibility complex class II (MHC II) and costimulatory molecules on the surface of BMDCs stimulated by PSC-ELVs. The treated BMDCs were incubated with the following fluorescently labeled antibodies at 4 °C for 30 min: BV421-labeled anti-CD11b, FITC-labeled anti-CD11c, PE-labeled anti-MHC II, APC-labeled anti-CD86, PE-Cy7-labeled anti-CD80, and PE-Cy7-labeled anti-CD40 (BioLegend, San Diego, CA, USA). An LSRFortessa X-20 instrument (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo software (Tree Star Inc., Ashland, OR, USA) were used to acquire and analyze the flow cytometry data.