Cd3e percp cy5

Cells were prestained with a 1:100 dilution of Zombie Aqua (Fixable Viability Dye; BioLegend, London, UK) for 20 minutes at 4°C in the dark. After 10 minutes, an equal volume of a 1:100 dilution of TruStain FcX PLUS (anti-mouse CD16/32 antibody) and True-Stain Monocyte Blocker (BioLegend) was added. After a washing step, cells were stained with CD3e-PerCP-Cy5.5, CD19-PerCP-Cy5.5 (eBioscience, Thermo Fisher Scientific), NK1.1-PerCP-Cy5.5, CD103-PerCP-Cy5.5, F4/80-FITC, Ly6G-BV785, Ly6C-BV650 (BioLegend), SiglecF-PerCP-Cy5.5, CD45-APC-Cy7, CD11b-PE-Cy7 and Tim4-PE (BD Biosciences, Erembodegem, Belgium) for 20 minutes at 4°C in the dark. Cells were analyzed with a BD FACSAria Fusion flow cytometer (BD Biosciences) and FlowJo software (FlowJo LLC, BD Biosciences), and gated first as live CD45⁺ single cells. Subsequently, CD3e⁺, CD19⁺, NK1.1⁺, CD103⁺ and SiglecF⁺ cells were eliminated, and CD11b⁺Ly6C⁺Ly6G^- monocytes, CD11b⁺Ly6C^-F4/80⁺Tim4⁺ Kupffer cells (KCs) and CD11b⁺Ly6C^-F4/80⁺Tim4^- monocyte-derived macrophages (MoMfs) were gated.

Blood, bone marrow, BAL, and lung cells were counted using an automated cell counter (Nexcelcom Bioscience, Lawrence, MA). Cells were incubated with Fc block (anti-mouse CD16/CD32; eBioscience) in 100 μl of FACS buffer (PBS, 0.5 % EDTA) followed by the antibody panels below. Labeled cells were analyzed using BD FACSCanto RUO (BD Biosciences, San Jose, CA) and the FlowJo data analysis software (Ashland, OR). Experiments represent an average of n = 5/group with up to 3 replicates. Antibodies used included: anti-mouse CD62L-APC, ICAM1-FITC, Ly6G-FITC, Ly6G-PerCP/Cy5.5, LFA-1-PE, CD45-APC/Cy7, CD4-PE, CD8-PB, CXCR2-PerCP/Cy5.5, Gr1-PB, Ly6C-PB (BioLegend, San Diego, CA), CD11C-APC, CD11b-PECy7, CD3e PE/Cy7, CD3e-PerCP/Cy5.5, CD71-PE, F4/80-APC (eBioscience).

Spleens were isolated 17 days after inoculation and mashed using 70 μm filters. Splenocytes were harvested after depletion of RBCs by the hypotonic lysis buffer (BD Biosciences). CD8α-positive splenocytes were isolated by positive selection with anti-CD8α microbeads (Miltenyi Biotec) according to the manufacturer's protocol. The purity of CD8α+ T cells were analyzed with a combination of antibodies specific for CD8α-FITC and CD3e-PerCP-cy5.5 (all purchased from eBioscience, San Diego, CA, USA) by flow cytometry. Then, 2×10⁶ CD8α-positive splenocytes were co-cultured with prepared 5×10⁵ BM-DCs pulsed with 30 μg/ml murine GPC3 proteins (Sino Biological, Inc.) for restimulation. Seven days later, the suspensions containing 4×10⁵ CD8α+ T cells in T-cell medium were added to each well in ELISPOT 96-well plates and stimulated in triplicate with 30 μg/ml GPC3 protein at 37°C for 24 h. The detection of GPC3-specific T cells producing IFN-γ was performed using an ELISPOT kit (BD Biosciences) according to the manufacturer's protocol. Lastly, the plates were dried at normal temperature and the spots were counted with an ELISPOT Reader (CTL Limited) by capturing the images of individual wells. Spot-forming cells were defined as the average number of spots per 4×10⁵ CD8α+ T cells from triplicate wells.