Pan dc enrichment kit

Manufactured by Miltenyi Biotec

Sourced in Germany

The Pan-DC Enrichment Kit is a laboratory product designed to isolate and enrich dendritic cells from a variety of cell sources. It provides a reliable and efficient method for the separation and purification of this cell population.

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Lab products found in correlation

Anti human cd3 fitc, by BD (2 mentions) Apc cy7 anti human hla dr, by BioLegend (2 mentions) Bv421 anti human cd11c, by BioLegend (2 mentions) Ficoll, by GE Healthcare (1 mentions) Anti human lineage cocktail, by BioLegend (1 mentions) Moflo astrios eq cell sorter, by Beckman Coulter (1 mentions) Facsaria, by BD (1 mentions) Easysep human pan dc pre enrichment kit, by STEMCELL (1 mentions) Golgistop, by BD (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Ficoll pacque plus, by GE Healthcare (1 mentions) Blood cell culture dna mini kit, by Qiagen (1 mentions) Dmxaa, by Selleck Chemicals (1 mentions) Cgamp, by InvivoGen (1 mentions) Tepp 46, by Selleck Chemicals (1 mentions) Collagenase d, by Roche (1 mentions) Pan t cell isolation kit, by Miltenyi Biotec (1 mentions) Anti human cd3, by Thermo Fisher Scientific (1 mentions) Pe anti human cd123, by BD (1 mentions) Human t cell nucleofection kit, by Lonza (1 mentions)

Spelling variants of this product

Human pan-DC enrichment kit (2 citations)

12 protocols using pan dc enrichment kit

Isolation and Enrichment of Human DCs

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PBMC from healthy human donors (Interregionale Blutspende SRK) were isolated using Ficoll (GE Healthcare, Chicago, IL, USA) density gradient centrifugation. Monocytes were enriched using the EasySep^TM Human Monocyte Enrichment Kit w/o CD16 Depletion (STEMCELL Technologies).
DC were isolated using the MACS-based Pan-DC Enrichment Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) followed by Fc-receptor blocking with anti-mouse CD16/32 (2.4G2, generated in house) for 15 min. Subsequently, cell surface markers were stained using anti-CD11c (3.9), anti-CD123 (6H6), anti-human lineage cocktail (CD3/14/16/19/20/56), anti-CD141 (M80), anti-HLA-DR (L243) (Biolegend, San Diego, CA, USA) in FACS buffer (PBS with 2% FBS and 1 mM EDTA) for 45 min on ice. Cells were then purified by FACS using the Moflo Astrios EQ cell sorter (Beckman Coulter, Nyon, Switzerland). Ethical approval provided by the Kantonale Ethikkommission Bern (KEK), Switzerland. Code: 2017-02246.

Kremenovic M., Rombini N., Chan A.A., Gruber T., Bäriswyl L., Lee D.J, & Schenk M. (2020). Characterization of a Myeloid Activation Signature That Correlates with Survival in Melanoma Patients. Cancers, 12(6), 1431.

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Single-cell RNA-seq of human dendritic cells

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Cells were sorted on a FACSAria (BD biosciences). For SmartSeq2 scRNAseq, single cells were index sorted using the indicated gating strategy, from FT7 cultures differentiated on OP9+OP_DLL1 for 18 days. For the 10X Genomics scRNAseq experiments from cultures, cells were harvested by pipetting, filtered through a 30 μm nylon mesh, and sorted by flow cytometry as live (LIVE/DEAD Fixable Aqua Dead^-) total hematopoietic (CD45⁺) or Lin^- HLA-DR⁺, using PE-CD45, or FITC-lineage cocktail (CD3, CD14, CD16, CD19, CD20, CD56) with BV786-HLA-DR, APC-BDCA2/CD303 and V450-CD11c, before loading into the Chromium Single Cell Controller apparatus. For the 10X Genomics scRNAseq experiment with cells enriched from adult PBMCs, cells from a healthy blood donor were separated by density gradient centrifugation, enriched by magnetic bead-based sorting using the pan-DC enrichment kit from Miltenyi, stained and sorted for Lin^- HLA-DR⁺ cells as done for the cells from cultures, before loading into the Chromium Single Cell Controller apparatus. Peripheral blood XCR1^- and XCR1⁺ cDC1 subsets were sorted from PBMCs enriched with the EasySep Human Pan-DC Pre-Enrichment Kit (stem cell technologies), by gating on Lin^- HLA-DR⁺ CD123^- CD45RA^- CD11c⁺ CD1c^- SIRPa^- CD141⁺ CLEC9A⁺ CADM1⁺ cells. Sorted cells were cultured for 14 days on OP9_DLL1 by plating 10,000 to 30,000 cells/ml with the FT7+G cytokine cocktail.

Balan S., Arnold-Schrauf C., Abbas A., Couespel N., Savoret J., Imperatore F., Villani A.C., Vu Manh T.P., Bhardwaj N, & Dalod M. (2018). Large-Scale Human Dendritic Cell Differentiation Revealing Notch-Dependent Lineage Bifurcation and Heterogeneity. Cell Reports, 24(7), 1902-1915.e6.

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Isolation of NSCLC Immune Cells

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Human NSCLC tissues and paracancerous tissues were obtained from patients with NSCLC at Shanghai Chest Hospital, Shanghai Jiao Tong University School of Medicine. Fresh tumor tissues and paracancerous tissues were digested with collagenase VIII and DNase I. Immune cells were enriched by density-gradient centrifugation. DCs were isolated using a Pan-DC Enrichment Kit (130-100-777, Miltenyi) for further experiments.

Hu Z., Yu X., Ding R., Liu B., Gu C., Pan X.W., Han Q., Zhang Y., Wan J., Cui X.G., Sun J, & Zou Q. (2023). Glycolysis drives STING signaling to facilitate dendritic cell antitumor function. The Journal of Clinical Investigation, 133(7), e166031.

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Neuroblastoma Activation of DCs

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DCs were purified from PBMC obtained from 3 healthy volunteers, with Pan-DC Enrichment kit (Miltenyi Biotec) and cocultured for 24 hours with neuroblastoma cell lines, in 24-well plate (1:1 ratio, 0.5 × 10⁶ of each cell types) in DMEM 10% FCS (2 mL), with or without R848 (1 μg/mL). CCL19 and CCL22 productions were measured by ELISA (Sigma-Aldrich) in the harvested supernatants.

Kacher J., Manches O., Aspord C., Sartelet H, & Chaperot L. (2022). Impaired Antitumor Immune Response in MYCN-amplified Neuroblastoma Is Associated with Lack of CCL2 Secretion and Poor Dendritic Cell Recruitment. Cancer Research Communications, 2(7), 577-589.

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Purification and Stimulation of Human Blood Pan-DCs

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Human blood pan-DCs were purified from PBMCs (isolated from healthy human donor fresh blood from Medcor Cambridge internal donor program, or Bioreclamation) with a pan-DC enrichment kit (Miltenyi Biotec). Cells were plated in 96-well round bottom plates at 45 000–50 000 cells per well, in the presence of 25 μg/ml sabatolimab antibody or hIgG4 isotype control, for 16–24 hours. Cells were stimulated by adding 1 μg/ml LPS for 6 hours, with GolgiStop (BD Biosciences) added for the last 4 hours. Cells were then stained with antibodies for surface markers and intracellular cytokines, and analyzed by flow cytometry. cDC2 subtype cells (marked as HLADR⁺ CD11c⁺ CD1c⁺ cells) made up the majority of cells and showed the most robust results, and hence were focused on in the analysis.

Schwartz S., Patel N., Longmire T., Jayaraman P., Jiang X., Lu H., Baker L., Velez J., Ramesh R., Wavreille A.S., Verneret M., Fan H., Hu T., Xu F., Taraszka J., Pelletier M., Miyashiro J., Rinne M., Dranoff G., Sabatos-Peyton C, & Cremasco V. (2022). Characterization of sabatolimab, a novel immunotherapy with immuno-myeloid activity directed against TIM-3 receptor. Immunotherapy Advances, 2(1), ltac019.

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Isolation of DCs and PBLs from Peripheral Blood

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Peripheral blood from healthy donors was obtained from buffy coats provided by the Red Cross donor center (Red Cross-Flanders, Mechelen, Belgium), and peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Ficoll Pacque PLUS, GE Healthcare, Amsterdam, The Netherlands). The Pan-DC enrichment kit (Miltenyi biotech) was used to isolate DCs from the PBMC. From the remaining PBMC fraction, peripheral blood lymphocytes (PBLs) were depleted from CD14+ monocytes using CD14+ immunomagnetic selection (CD14 Reagent, Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. The CD14-depleted cell fraction (i.e., peripheral blood lymphocytes (PBLs)) was cryopreserved in FBS supplemented with 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich, Bornem, Belgium) and stored at −80 °C for later use in an allogeneic mixed leukocyte reaction (allo-MLR).

Meena M., Van Delen M., De Laere M., Sterkens A., Costas Romero C., Berneman Z, & Cools N. (2021). Transmigration across a Steady-State Blood–Brain Barrie Induces Activation of Circulating Dendritic Cells Partly Mediated by Actin Cytoskeletal Reorganization. Membranes, 11(9), 700.

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Isolation and Stimulation of BMDCs

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BMDCs were harvested for experiments in vitro. For further analysis, BMDCs were counted and seeded in cell culture plates followed by cGAMP or purified Tu-DNA stimulation, respectively. cGAMP and Tu-DNA were transfected using Tenfect DNA transfection reagent (TEYE Corporation) according to the manufacturer’s instructions. Tu-DNA was obtained from MC38 genomic DNA using a Blood & Cell Culture DNA Mini Kit (13323, QIAGEN). cGAMP was purchased from InvivoGen Inc. TEPP-46 and DMXAA were purchased from Selleck. Sodium oxamic acid was purchased from Adamas-beta. DCA and cyclodextrin were purchased from Sigma-Aldrich. Human DCs were freshly isolated from NSCLC tissues and paracancerous tissues and purified with a Pan-DC Enrichment Kit (130-100-777, Miltenyi). The purified human DCs were untreated or treated with 2-DG or TEPP-46 for 8 hours and then used for further experiments.

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Isolation of Murine Hepatic Dendritic Cells

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For hepatic DC isolation, Balb/c (WT) mice were anaesthetized with isoflurane (induction 4%, maintenance 2%). After the portal vein was cannulated, the liver was perfused with cold RPMI 1640 containing 0.1% (w/v) collagenase D (Roche Diagnostics, Indianapolis, IN) before harvesting. Each liver was then gently minced and digested with 0.1% collagenase D in RPMI 1640 for 30 min at 37°C prior to passage through a 70-μm cell strainer, Percoll gradient centrifugation and separation with αCD11c+/αPDCA-1+ microbeads (Pan-DC Enrichment Kit, Miltenyi Biotec, Teterow, German).

Liu Y.J., Li K., Yang L., Tang S.T., Wang X.X., Cao G.Q., Li S., Lei H.Y, & Zhang X. (2015). Dendritic Cells Regulate Treg-Th17 Axis in Obstructive Phase of Bile Duct Injury in Murine Biliary Atresia. PLoS ONE, 10(9), e0136214.

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Immunomodulation of Immune Cells by Compounds

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Human macrophages, DC and T cells were isolated from peripheral blood mononuclear cells (PBMC). Therefore, PBMC were purified from fully anonymized leukapheresis reduction chambers, obtained from the blood bank with the informed consent of healthy donors by Ficoll gradient centrifugation according to standard methods (Ficoll reagent was purchased from Merck). Total macrophages, DC or T cells were selected using CD14 microbeads (macrophages) or negatively enriched using the Pan‐DC Enrichment Kit or the Pan T Cell Isolation Kit (Miltenyi Biotech). All experiments were carried out according to the declaration of Helsinki and were approved by the ethical committee of the University of Münster Medical School (2008‐180‐f‐S). After isolation, human macrophages or DC were activated for 12 h with LPS (10 μg/mL) and cultured for additional 48 h in the presence of compounds (±)‐4, 4 and ent‐4 at indicated concentrations or PBS. Human T cells were activated with plate‐bound anti‐CD3 and soluble anti‐CD28 (clones UCHT1 and CD28.2; 1 μg/mL each antibody, both purchased from Biolegend, San Diego, CA). Finally, cells were subjected to flow cytometry.

Martin B., Schepmann D., Bernal F.A., Schmidt T.J., Che T., Loser K, & Wünsch B. (2020). Enantiomerically Pure Quinoline‐Based κ‐Opioid Receptor Agonists: Chemoenzymatic Synthesis and Pharmacological Evaluation. Chemmedchem, 15(15), 1408-1420.

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Dendritic Cell Enrichment from PBMCs

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DCs were enriched from freshly isolated PBMCs using the Miltenyi PanDC Enrichment Kit (#130-100-777) according to the manufacturer’s protocol and 2× enrichment was performed for all experiments.

He M., Soni B., Schwalie P.C., Hüsser T., Waltzinger C., De Silva D., Prinz Y., Krümpelmann L., Calabro S., Matos I., Trumpfheller C., Bacac M., Umaña P., Levesque M.P., Dummer R., van den Broek M, & Gasser S. (2022). Combinations of Toll-like receptor 8 agonist TL8-506 activate human tumor-derived dendritic cells. Journal for Immunotherapy of Cancer, 10(6), e004268.

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