Fitc anti mouse cd11c

HI rWR (A3 or F17 where indicated) or UV-attenuated WR-mini-OVA was used to infect either MutuDCs, 293A cells, or 293KbC2 cells (57 (link)) for 2 h with rocking at an MOI of 10 before the inoculum was replaced with IMDM-10. Splenocytes from a naive OT-I mouse were harvested and subjected to CD8α-negative enrichment (Miltenyi Biotech, 130-096-543). The eEnrichment efficiency was checked by flow cytometry, and samples were at least 85% CD8α⁺ Vα2⁺ T cells. OT-I CD8⁺ T cells were added to V-bottom plates in D10 with β-mercaptoethanol, and infected cells were added in the same medium at a ratio of 1:2 (infected cell target to OT-I). After 24 h, the cells were labeled with anti-mouse CD11c-FITC (BioLegend, clone N418), anti-mouse CD8α-PE-Cy7 (BioLegend, clone 53.67), anti-mouse TCR Vα2-APC (BioLegend, clone B20.1), anti-mouse CD69-PerCP-Cy5.5 (BioLegend, clone H1.2F3), and anti-mouse CD25 (BioLegend, clone 3C7) antibodies diluted in PBS with 2% FBS. Events were gated sequentially on SSC-A × FSC-A (lymphocytes), FSC-H × FSC-W (single cells), SSC-H x SSC-W (single cells), SSC-A × FITC/GFP^– (MutuDC exclusion), PE-Cy7 × APC (CD8⁺ Vα2⁺ cells), and PE × PerCP-Cy5.5 (CD25⁺ CD69⁺ activated cells) to determine the percentages of CD8⁺ Vα2⁺ T cells that upregulate activation markers.

Mouse spleens were collected at 7 days post immunization, embedded in OCT medium and frozen at −80 °C before processing into 5 μm sections. Samples were fixed in cold 4% paraformaldehyde for 10 min followed by blocking in 5% FBS for 1 h at room temperature. For GC analysis, Alexa-Fluor 594 B220 (Biolegend 103254), Alexa-Fluor 488 anti-mouse IgG (Biolegend 405319), and Alexa-Fluor 647 anti-GL-7 (Biolegend 144605) or for macrophage depletion, Alexa-Fluor 594 B220, FITC anti-mouse CD68 (Biolegend 137005) /FITC anti-mouse CD11c (Biolegend 117305), and Alexa-Fluor 647 anti-mouse CD169 (Biolegend 142407) were incubated at 1:100 overnight at 4 °C. Samples were mounted on glass slides in ProLong Antifade reagent (Invitrogen). Images were acquired on a Zeiss LSM700 confocal microscope and on a Mirax Midi slide scanner system for whole organ imaging. GCs were defined as discrete areas of B220, GL-7, and IgG signal colocalization and counted manually in each spleen section. PBs were defined as IgG⁺ cells and quantified in two 1 mm² areas in each spleen section using ImageJ.

To evaluate DCs maturation, CT26 tumor-bearing mice (110–130 mm³) were divided into six groups, which were treated with Saline, Gd-NCPs ([Gd³⁺] = 30 mg kg⁻¹), H@Gd-NCPs ([Gd³⁺] = 30 mg kg⁻¹ and [Hemin] = 12.5 mg kg⁻¹) with radiation (6 Gy × 1) and Saline, Gd-NCPs, H@Gd-NCPs without radiation, respectively. Five days post radiation, mice were sacrificed and tumor-draining lymph nodes (TDLNs) were harvested for flow cytometry analysis. The TDLNs were ground into single-cell suspension, stained with FITC anti-mouse CD11c (0.25 µg per million cells in 100 µL volume), PE anti-mouse CD86 (0.25 µg per million cells in 100 µL volume), APC anti-mouse CD80 (1.0 µg per million cells in 100 µL volume) antibodies (BioLegend, America) and then detected by flow cytometry (BD Calibur).

For surface staining, the cell suspension was incubated with fluorescently labeled antibody at room temperature for 20 minutes. For CD206 staining, the samples were fixed and permeabilised with BD Cytofix/Cytoperm Fixation/Permeabilization Solution kit (BD Biosciences, US), and then incubated with fluorescently labeled antibody at 4 °C for 35 minutes. For intracellular TNF-α staining, the samples were stimulated by LPS (100 ng/ml, Beyotime) for 4 h, fixed and permeabilised, and then incubated with fluorescently labeled antibody at 4 °C for 35 minutes. Bacteria were stained using the BacLight™ Red kit (Invitrogen, B-35001). miRNA was labelled with fluorescein amidites (FAM). The monoclonal antibodies used were as follows: APC-Cy7-anti-Mouse F4/80, Pacific Blue-anti-Mouse CD11b, FITC-anti-Mouse CD11c, APC-anti-Mouse CD206, APC-Cy7-anti-Mouse TCRβ, APC-anti-Mouse NK1.1, PerCP-Cy5-5-anti-Mouse CD45, PE-Cy7-anti-Mouse CD3, PE-anti-Mouse CD4, FITC-anti-Mouse CD8, APC-anti-Mouse TNF-α, all purchased from Biolegend; FITC-anti-Human CD86 (20 μl/ test), APC-anti-Human CD206 (20 μl/test), purchased from BD Pharmingen. Flow cytometry or Image Stream was conducted using a BD FACS CantoII or Millipore ISX with fluorochrome-conjugated cells, and the data was analysed using FlowJo software version 10.4 or IDEAS version 6.0.