Spleens were collected from EAE mice and dissociated through 70 μm cell strainer to prepare single cell suspensions. After treatment with RBC lysis buffer (Biolegend, CA, USA) cells were extensively washed with complete IMDM by centrifugation at 1300 rpm for 5 minutes at 4°C. Splenic CD11c+ DCs were isolated by positive selection with magnetic beads (CD11c+ cell isolation kit, Miltenyi Biotec, CA, USA) and seeded in a 96-well U-bottom plate at a concentration of 20,000 DCs/well. Bead isolated MOG35-55-reactive naïve CD4+ T cells were seeded over DCs at 200,000 T cells/well. Additionally, MOG35-55 peptide was added to cultures at a 25 μg/mL concentration. Cultures were incubated for 72 h at 37°C, and dye decay was analyzed by flow cytometry in CD4+ T cells.
Cd11c cell isolation kit
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
The CD11c+ cell isolation kit is a tool designed to isolate CD11c+ cells from a mixed cell population. It utilizes magnetic bead-based separation technology to selectively capture and isolate the target cell type. The core function of this product is to enable the purification of CD11c+ cells for downstream applications, such as cell-based research or therapeutic development.
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5 protocols using cd11c cell isolation kit
1
Antigen-Specific CD4+ T Cell Activation Assay
2
Splenic CD11c+ DC Isolation and T Cell Activation Assay
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Splenic CD11c+ DCs were isolated from spleens from MOG35–55-tolerized and non-tolerized EAE mice using magnetic beads (CD11c+ cell isolation kit, Miltenyi Biotec, CA, USA). Isolated DCs were surface stained with antibodies against CD11c, CD11b, and CD103 for 20 min at 4°C and then sorted into four CD11c+ populations: CD11b−CD103−, CD11b+CD103−, CD11b+CD103+, and CD11bhiCD103+. Sorting was performed on FACSAria III (BD Biosciences, CA, USA). Sorted DCs were plated in U-bottom 96 well plates at a density of 20,000 cells/well. For Ag-presentation assays, naive CD4+ T cells from WT mice were isolated using magnetic beads (Naive CD4+ T cell isolation kit, Miltenyi Biotec, CA, USA). Isolated T cells were CD3+CD4+CD44−CD62L+ as analyzed by flow cytometry. 200,000 naive CD4+ T cells were added to each well of the cell culture plate containing DCs (ratio of 1 DC: 10 T cells) and plates were incubated at 37°C in the presence of anti-CD3 (0.5 µg/mL). Cells were collected after 72 h and analyzed by flow cytometry, while cytokine concentrations in culture supernatants were measured by ELISA.
3
Murine Bone Marrow-Derived Dendritic Cell Isolation
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The bone marrow-derived mononuclear cells (BMMCs) were prepared from C57BL/6 mice tibia and femur suspensions by depletion of red blood cells and cultured at a density of 2×106 cells/ml in the RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 10 ng/mL recombinant mouse granulocyte macrophage colony stimulating factor (rmGM-CSF; PeproTech, Rocky Hill, NJ, USA) and 10 ng/mL rmIL-4 (PeproTech) at 37 °C with 5% CO2 for 6 days. On day 6, the imDCs were isolated by negative selection using magnetic beads (CD11c Cell Isolation Kit, Miltenyi biotech, Germany) following the manufacturer indications. In addition, cells were fixed and allowed to adhere to poly-L-lysine coated coverslips before preparation for scanning electron microscopy observation.
4
Assessing CD11c+ DC-Driven CD8+ T Cell Activation
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The capacity of CD11c + DCs to activate SIINFEKL specific T CD8+ cells in vivo was assessed. On day three after PLGA MS OVA immu nization, spleens were collected and CD11c positive DCs were enriched by positive selection using a CD11c + Cell Isolation Kit (Miltenyi Biotec, Germany). Subsequently, OT I recipient mice were immunized by intravenously transfer of 4 Â 10 6 sorted CD11c + cells. For some experiments sorted CD11c + cells were externally pulsed with 1 Â 10 6 M SIINFEKL peptide for 1 h at 37 °C before adoptive transfer. Five days after CD11c + transfer, spleens from OT I mice were removed and splenocytes were ana lyzed for SIINFEKL specific T CD8+ generation by performing an intracellular cytokine staining (ICS) for IFN c.
5
Mechanisms of Impaired CD8+ T Cell Priming
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In order to gain mechanistic insights into the cause of the com promised ability of chronically stressed mice to prime SIINFEKL specific T CD8+ cells, we examined the overall capacity of isolated CD11c + cells to prime an autologous T CD8+ line established from OT I mice infected with a recombinant vaccinia virus (rVV) expressing SIINFEKL. Spleen cells were collected from control and SDR mice on the last day of the stress procedure. Subsequently, CD11c + cells were enriched by positive selection using a CD11c + Cell Isolation Kit (Miltenyi Biotec, Germany). CD11c + DCs were incubated with PLGA MS OVA/CpG at 37 °C overnight, to allow uptake and processing of encapsulated OVA peptide. Cells were collected the next day and used as stimulator cells in an antigen presentation assay. A SIINFEKL/H 2K b specific CTL line was gener ated from OT I mice which had been infected with rVV SIINFEKL as previously described (Basler et al., 2006) and used in the antigen presentation assay at an effector to stimulator (E:S) ratio of 1:2 and 1:1. In a second approach, CD11c + DCs were pulsed with 10 À7 M of SIINFEKL peptide for 1 h at 37 °C, washed three times with PBS and used as stimulator cells. Direct antigen presentation was measured using IFN c ICS.
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