Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Muccioli, Maria; Pate, Michelle; Omosebi, Omowaleola; Benencia, Fabián

doi:10.3791/2785-v

Cited by 3 publications

(4 citation statements)

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“…Primary Cell Isolation and Culture: Bone marrow-derived dendritic cells (BMDCs) were prepared according to the published procedure. [54,55] In brief, tibias and femurs were dissected and the attached tissues were removed carefully. The inside of a bone was rinsed to a sterile Petri dish with 5 mL of RPMI 1640 medium without serum but with 2% penicillin and streptomycin (GIBCO).…”

Section: Methodsmentioning

confidence: 99%

Arf1 Ablation in Colorectal Cancer Cells Activates a Super Signal Complex in DC to Enhance Anti‐Tumor Immunity

Ma,

Fang,

et al. 2023

Advanced Science

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The anti‐tumor immune response relies on interactions among tumor cells and immune cells. However, the molecular mechanisms by which tumor cells regulate DCs as well as DCs regulate T cells remain enigmatic. Here, the authors identify a super signaling complex in DCs that mediates the Arf1‐ablation‐induced anti‐tumor immunity. They find that the Arf1‐ablated tumor cells release OxLDL, HMGB1, and genomic DNA, which together bound to a coreceptor complex of CD36/TLR2/TLR6 on DC surface. The complex then is internalized into the Rab7‐marked endosome in DCs, and further joined by components of the NF‐κB, NLRP3 inflammasome and cGAS‐STING triple pathways to form a super signal complex for producing different cytokines, which together promote CD8+ T cell tumor infiltration, cross‐priming and stemness. Blockage of the HMGB1‐gDNA complex or reducing expression in each member of the coreceptors or the cGAS/STING pathway prevents production of the cytokines. Moreover, depletion of the type I IFNs and IL‐1β cytokines abrogate tumor regression in mice bearing the Arf1‐ablated tumor cells. These findings reveal a new molecular mechanism by which dying tumor cells releasing several factors to activate the triple pathways in DC for producing multiple cytokines to simultaneously promote DC activation, T cell infiltration, cross‐priming and stemness.

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Section: Methodsmentioning

confidence: 99%

Arf1 Ablation in Colorectal Cancer Cells Activates a Super Signal Complex in DC to Enhance Anti‐Tumor Immunity

Ma,

Fang,

et al. 2023

Advanced Science

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“…Cell culture: DCs were generated through the culture of bone marrow cells isolated from femurs of C57BL/6 mice (six-eight-week-old) in complete medium (Gibco, USA) as previously described. [87] Cells were cultured in complete medium added with murine GM-CSF and IL-4 (20 ng mL −1 , Pepro Tech, USA) and refreshed every two days. On day 6, the cells were harvested for further analysis.…”

Section: Methodsmentioning

confidence: 99%

“…[14] A specific segment of MBP consisting of amino acids 87-99 (MBP 87-99 ) has been shown to be a major target of T cells in neuroinflammation. Previous studies have demonstrated that MBP 87-99 A 91 , an APL of MBP [87][88][89][90][91][92][93][94][95][96][97][98][99] in which the lysine residue at position 91 is replaced with alanine, can alter the polarity of inflammatory CD4 + T cells and induce neuroprotective effect. [15,16] However, the clinical application of APLs, such as MBP 87-99 A 91 , comes with many challenges, including poor stability due to enzymatic degradation in blood circulation [17] and damaging biological barriers that result in toxicity to tissues and organs.…”

Section: Introductionmentioning

confidence: 99%

Small Extracellular Vesicles Derived from Altered Peptide Ligand‐Loaded Dendritic Cell Act as A Therapeutic Vaccine for Spinal Cord Injury Through Eliciting CD4⁺ T cell‐Mediated Neuroprotective Immunity

Wang,

Li,

Liang

et al. 2023

Advanced Science

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The balance among different CD4+ T cell subsets is crucial for repairing the injured spinal cord. Dendritic cell (DC)‐derived small extracellular vesicles (DsEVs) effectively activate T‐cell immunity. Altered peptide ligands (APLs), derived from myelin basic protein (MBP), have been shown to affect CD4+ T cell subsets and reduce neuroinflammation levels. However, the application of APLs is challenging because of their poor stability and associated side effects. Herein, it is demonstrate that DsEVs can act as carriers for APL MBP87‐99A91 (A91‐DsEVs) to induce the activation of 2 helper T (Th2) and regulatory T (Treg) cells for spinal cord injury (SCI) in mice. These stimulated CD4+ T cells can efficiently “home” to the lesion area and establish a beneficial microenvironment through inducing the activation of M2 macrophages/microglia, inhibiting the expression of inflammatory cytokines, and increasing the release of neurotrophic factors. The microenvironment mediated by A91‐DsEVs may enhance axon regrowth, protect neurons, and promote remyelination, which may support the recovery of motor function in the SCI model mice. In conclusion, using A91‐DsEVs as a therapeutic vaccine may help induce neuroprotective immunity in the treatment of SCI.

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“…In this case, the differently treated cells need to be differentially labeled to be able to distinguish them later in vivo. While the range of cytoplasmic staining is limited, additional differential labeling can be achieved by feeding the cells Qdot nanocrystals [14]. Crucially, however, different treatment and thus different activation states may affect migration to the lymph node or intra‐nodal distribution after adoptive transfer, and must be carefully controlled. Continue with cell staining protocol (see below). After staining is complete, resuspend the pellet in sterile HBSS for subsequent subcutaneous injection at a concentration of 2 × 10 6 cells per 25–50 μl (40‐80 × 10 6 /ml).…”

Section: Intravital Imaging Of Dc‐t Cell Interactions In Mouse Periph...mentioning

confidence: 99%

Guidelines for visualization and analysis of DC in tissues using multiparameter fluorescence microscopy imaging methods

et al. 2023

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This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state‐of‐the‐art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non‐lymphoid tissues. Here, we provide detailed procedures for a variety of multiparameter fluorescence microscopy imaging methods to explore the spatial organization of DC in tissues and to dissect how DC migrate, communicate, and mediate their multiple functional roles in immunity in a variety of tissue settings. The protocols presented here entail approaches to study DC dynamics and T cell cross‐talk by intravital microscopy, large‐scale visualization, identification, and quantitative analysis of DC subsets and their functions by multiparameter fluorescence microscopy of fixed tissue sections, and an approach to study DC interactions with tissue cells in a 3D cell culture model. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer‐reviewed by leading experts and approved by all co‐authors, making it an essential resource for basic and clinical DC immunologists.

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Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Cited by 3 publications

References 5 publications

Arf1 Ablation in Colorectal Cancer Cells Activates a Super Signal Complex in DC to Enhance Anti‐Tumor Immunity

Arf1 Ablation in Colorectal Cancer Cells Activates a Super Signal Complex in DC to Enhance Anti‐Tumor Immunity

Small Extracellular Vesicles Derived from Altered Peptide Ligand‐Loaded Dendritic Cell Act as A Therapeutic Vaccine for Spinal Cord Injury Through Eliciting CD4⁺ T cell‐Mediated Neuroprotective Immunity

Guidelines for visualization and analysis of DC in tissues using multiparameter fluorescence microscopy imaging methods

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Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Cited by 3 publications

References 5 publications

Arf1 Ablation in Colorectal Cancer Cells Activates a Super Signal Complex in DC to Enhance Anti‐Tumor Immunity

Arf1 Ablation in Colorectal Cancer Cells Activates a Super Signal Complex in DC to Enhance Anti‐Tumor Immunity

Small Extracellular Vesicles Derived from Altered Peptide Ligand‐Loaded Dendritic Cell Act as A Therapeutic Vaccine for Spinal Cord Injury Through Eliciting CD4+ T cell‐Mediated Neuroprotective Immunity

Guidelines for visualization and analysis of DC in tissues using multiparameter fluorescence microscopy imaging methods

Contact Info

Product

Resources

About

Small Extracellular Vesicles Derived from Altered Peptide Ligand‐Loaded Dendritic Cell Act as A Therapeutic Vaccine for Spinal Cord Injury Through Eliciting CD4⁺ T cell‐Mediated Neuroprotective Immunity