Cd8a fitc

CD4⁺ and CD8⁺ differentiation of T cells from stimulated splenocytes as similarly described above in lymphocyte proliferation were incubated with CD3e-PE, CD8a-FITC, and CD4-PerCPVio700 antibodies (Miltenyi-Biotec, Bergisch Gladbach, Germany) at 4 °C for 30 min in dark. Additionally, to compare the induction of MHCI and MHCII molecules, FITC labeled MHC class 1 (H-2Kb) and MHC class II (I-A/I-E) FACS analysis markers (Bio-Rad Laboratories, Inc., Hercules, CA, USA) were used according to the manufacturer’s protocol. Cells were washed and resuspended with MACSQuant running buffer (Miltenyi-Biotec, Bergisch Gladbach, Germany) before flow cytometric analysis. The percentage of CD3⁺CD4⁺, CD3⁺CD8⁺ T cells, MHCI, and MHCII was analyzed using a benchtop flow cytometer, MACSQuant^® analyzer (Miltenyi-Biotec, Bergisch Gladbach, Germany).
Moreover, stimulated splenocytes in 24-well plates were harvested and total RNA was isolated. cDNA was then synthesized using reverse transcription master premix (Elpis Biotech, Daejeon, Republic of Korea) with oligo d(T)15 primer according to the manufacturer’s protocol. Levels of IFN-γ, TNF-α, and IL-4 mRNA were analyzed by qPCR using the primers listed in Table 1. The relative cytokine expression levels were determined by the 2^−∆∆CT method using β-actin as the housekeeping gene.

mDCs and pDCs were detached from the plates with DPBS 1X (Thermo Fisher Scientific, Waltham, MA, USA) + 0.5 mM EDTA (Thermo Fisher Scientific, Waltham, MA, USA). Then, cells were washed with DPBS 1X + 0.5% BSA (Sigma-Aldrich, St. Louis, MO, USA) and labeled with CD8a FITC, Ly6C FITC, CD11c APC-Vio770, CD45R(B220) PE-Vio770, CD11c PE, MHCII APC, CD80 FITC, and 7-AAD Staining solution (Miltenyi Biotec, Bergisch Gladbach, Germany). Flow Cytometer acquisition was performed using NAVIOS (Beckman Coulter, Brea, CA, USA). Flow cytometer analysis was performed using Kaluza Software 1.5 (Beckman Coulter, Brea, CA, USA).