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Pan dendritic cell isolation kit

Manufactured by Miltenyi Biotec
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Sourced in Germany, United States

The Pan Dendritic Cell Isolation Kit is designed for the isolation of dendritic cells from various sources, such as peripheral blood mononuclear cells (PBMCs) or splenocytes. The kit utilizes a magnetic separation-based approach to enrich for dendritic cells.

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Lab products found in correlation

Celltrace violet dye, by Thermo Fisher Scientific (2 mentions) Ack lysis buffer, by Thermo Fisher Scientific (2 mentions) Anti qa 1b neutralizing antibody 6a8.6f10 1a16, by BD (2 mentions) Facs aria cell sorter flow cytometer, by BD (2 mentions) Macs ls separation column, by Miltenyi Biotec (2 mentions) Tcl buffer, by Qiagen (2 mentions) Violettrace, by Thermo Fisher Scientific (1 mentions) F4 80, by Thermo Fisher Scientific (1 mentions) Na ve cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Merck Group (1 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions) Naive cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions) Cd4 t cells isolation kit, by Miltenyi Biotec (1 mentions) Facsaria 2, by BD (1 mentions) Podoplanin, by BioLegend (1 mentions) Lyve 1, by Thermo Fisher Scientific (1 mentions) Lsm 780, by Zeiss (1 mentions) Zen imaging software, by Zeiss (1 mentions) Attune nxt, by Thermo Fisher Scientific (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)

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Mouse Pan Dendritic Cell Isolation Kit Cell isolation kits Isolation kits

26 protocols using pan dendritic cell isolation kit

1

Isolation and In Vitro Assay for Murine T-Cell Subsets

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Spleen and LN cells were harvested from WT or immunized mice. Single cell suspensions were prepared, and RBCs were lysed using Ack lysis buffer (ThermoFisher scientific A1049201). Total CD4+ (Miltenyi 130–049-201) and CD8+ (Miltenyi 130–049-401) T cells were positively purified using Miltenyi kits following established manufacturer protocol followed by FACS sorting. Similarly, antigen presenting cells were isolated using Miltenyi Pan Dendritic Cell (DC) Isolation Kit (Miltenyi 130–100-875). Post CD4+ T cell enrichment, cells were counted and labelled with CellTrace™ Violet Dye (ThermoFisher scientific C34557) according to manufacturer instructions. In vitro proliferation/suppression assays were set up according to previously published protocol47 (link). Briefly, labelled CD4+ T cells were co-cultured either with CD8+ T cells (1:1 ratio, 0.25X106 cells/well) or without CD8+ T cells in the presence of pan DC’s (0.75X106 cells/well). In some of the suppression experiments cells were pre-included with 10ug/ml of anti-Qa-1b neutralizing antibody (6A8.6F10.1A16, BD Biosciences)22 (link),48 (link). Cells were cultured in a total volume of 200 ul in a 96-well round bottom plate. The CD4+ T cells were stimulated with either MOG35–55 or not. On day 7, the cells were washed and stained with surface antibodies and analyzed on LSR II (Becton Dickinson).
Saligrama N., Zhao F., Sikora M.J., Serratelli W.S., Fernandes R.A., Louis D.M., Yao W., Ji X., Idoyaga J., Mahajan V.B., Steinmetz L.M., Chien Y.H., Hauser S.L., Oksenberg J.R., Garcia K.C, & Davis M.M. (2019). Opposing T Cell Responses in Experimental Autoimmune Encephalomyelitis. Nature, 572(7770), 481-487.
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2

Isolation and In Vitro Assay for Murine T-Cell Subsets

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Spleen and LN cells were harvested from WT or immunized mice. Single cell suspensions were prepared, and RBCs were lysed using Ack lysis buffer (ThermoFisher scientific A1049201). Total CD4+ (Miltenyi 130–049-201) and CD8+ (Miltenyi 130–049-401) T cells were positively purified using Miltenyi kits following established manufacturer protocol followed by FACS sorting. Similarly, antigen presenting cells were isolated using Miltenyi Pan Dendritic Cell (DC) Isolation Kit (Miltenyi 130–100-875). Post CD4+ T cell enrichment, cells were counted and labelled with CellTrace™ Violet Dye (ThermoFisher scientific C34557) according to manufacturer instructions. In vitro proliferation/suppression assays were set up according to previously published protocol47 (link). Briefly, labelled CD4+ T cells were co-cultured either with CD8+ T cells (1:1 ratio, 0.25X106 cells/well) or without CD8+ T cells in the presence of pan DC’s (0.75X106 cells/well). In some of the suppression experiments cells were pre-included with 10ug/ml of anti-Qa-1b neutralizing antibody (6A8.6F10.1A16, BD Biosciences)22 (link),48 (link). Cells were cultured in a total volume of 200 ul in a 96-well round bottom plate. The CD4+ T cells were stimulated with either MOG35–55 or not. On day 7, the cells were washed and stained with surface antibodies and analyzed on LSR II (Becton Dickinson).
Saligrama N., Zhao F., Sikora M.J., Serratelli W.S., Fernandes R.A., Louis D.M., Yao W., Ji X., Idoyaga J., Mahajan V.B., Steinmetz L.M., Chien Y.H., Hauser S.L., Oksenberg J.R., Garcia K.C, & Davis M.M. (2019). Opposing T Cell Responses in Experimental Autoimmune Encephalomyelitis. Nature, 572(7770), 481-487.
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3

Antigen-Specific T Cell Proliferation Assay

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Dendritic cells were purified from spleens and mesenteric lymph nodes of WT mice injected with 1 × 106 B16-FLT3L cells using the Pan Dendritic Cell (DC) Isolation Kit (Miltenyi, Germany). Naïve CD4+ T cells were purified following manufacturer’s instructions (Miltenyi, Germany) from the spleen and mesenteric lymph nodes of OT-II mice and labelled with 3 μM VioletTrace (ThermoFisher). Purity was always greater than 98% for both purified DCs and T cells. 1 ×105 DCs were incubated with peptide (50 nM) for 1 h before adding 1 *105 T cells. Proliferation was assessed using dilution of VioletTrace and analyzed by flow cytometry after 3.5 days of coculture.
Cheloha R.W., Woodham A.W., Bousbaine D., Wang T., Liu S., Sidney J., Sette A., Gellman S.H, & Ploegh H.L. (2019). Recognition of MHC-II peptide ligands that contain β-amino acids. Journal of immunology (Baltimore, Md. : 1950), 203(6), 1619-1628.
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4

Corneal Injury Induces CD11c+ Cells

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We have previously reported that 2.0-mm circular incisions of the corneal epithelium induce the generation of CS cells that express the CD11c surface marker.20 (link) Corneas of BALB/c mice were trephined as described previously, and CD11c+ spleen cells were isolated 4 days later using a Miltenyl Biotec (Santa Barbara, CA) pan dendritic cell (DC) isolation kit.20 (link) The CD11c+ cells from trephined mice and untreated mice were used for detecting regulatory cell activity in vitro.20 (link)
Neelam S, & Niederkorn J.Y. (2020). Corneal Nerve Ablation Abolishes Ocular Immune Privilege by Downregulating CD103 on T Regulatory Cells. Investigative Ophthalmology & Visual Science, 61(4), 25.
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5

Isolation and Characterization of Adipocyte and Adipose Immune Cell Fractions

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Adipocyte and adipose SVF, ART and ATM fractions were isolated from epididymal VAT10 (link). Briefly, adipocytes were incubated with biotinylated CD45 antibody (eBiosciences, 13-0451-85, 1:400 dilution) to remove leukocyte contamination and the purified CD45 negative adipocytes were used for the gene expression analysis and cell co-culture. ATMs and ARTs were isolated from SVF with biotinylated F4/80 (eBiosciences, 13-4801-85, 1:200 dilution) and CD3e (eBiosciences, 13-0031-85, 1:100 dilution) antibodies, respectively. BMDMs and PMs were generated39 (link). DCs were isolated from spleens of WT and aMHCII−/− mice by using Miltenyi Pan Dendritic Cell Isolation Kit. Cell and tissue RNA samples were isolated and analysed, as previously described10 (link).
Deng T., Liu J., Deng Y., Minze L., Xiao X., Wright V., Yu R., Li X.C., Blaszczak A., Bergin S., DiSilvestro D., Judd R., Bradley D., Caligiuri M., Lyon C.J, & Hsueh W.A. (2017). Adipocyte adaptive immunity mediates diet-induced adipose inflammation and insulin resistance by decreasing adipose Treg cells. Nature Communications, 8, 15725.
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6

Sorting Immune and Stromal Cells from Murine Lymph Nodes

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10–14 week old C57BL/6 males served as gLN donors, and biological triplicates or quadruplicates were collected. Cells were sorted using a FACS Aria cell sorter flow cytometer (Becton Dickinson). MLN dendritic cells were pre-enriched using a Pan Dendritic Cell Isolation Kit (130–100-875, Miltenyi Biotec) and LS MACS Separation Columns (Miltenyi Biotec). Dendritic cells were sorted as Aqua–CD45+Lin–(CD3–B220–NK1.1–CD19–) CD11chi, and the subpopulations further as MHCIIhiCD103+CD11b– and MHCIIhiCD103+CD11b+. Stromal cells were not pre-enriched and sorted as Aqua–CD45–TER119–CD24–TCRβ–B220–CD11c– cells, and the subpopulations further as podoplanin+CD31– (FRCs) and podoplanin+CD31+(LECs). Three hundred cells were sorted directly into 25 μl TCL buffer (Qiagen, 1031576) supplemented with 1% β-mercaptoethanol at single cell precision. Samples were kept at room temperature for 5 min, spun down and kept at −80 °C until further processing.
Esterházy D., Canesso M.C., Mesin L., Muller P.A., de Castro T.B., Lockhart A., ElJalby M., Faria A.M, & Mucida D. (2019). Compartmentalized gut lymph node drainage dictates adaptive immune responses. Nature, 569(7754), 126-130.
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7

Isolation of Splenic DC, Treg, and Naive T Cells

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Splenic DC were isolated by negative immune-magnetic selection using the Pan Dendritic Cell Isolation Kit (Miltenyi Biotec). Splenic Treg and naive T cells were isolated using the CD4+CD25+ Treg isolation kit and naive CD4+ isolation kit, respectively. All cell isolations were performed as recommended by the manufacturer (Miltenyi Biotec).
Klaus T., Wilson A.S., Vicari E., Hadaschik E., Klein M., Helbich S.S., Kamenjarin N., Hodapp K., Schunke J., Haist M., Butsch F., Probst H.C., Enk A.H., Mahnke K., Waisman A., Bednarczyk M., Bros M., Bopp T, & Grabbe S. (2022). Impaired Treg-DC interactions contribute to autoimmunity in leukocyte adhesion deficiency type 1. JCI Insight, 7(24), e162580.
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8

DC-Mediated T Cell Activation by Bacterial Extracts

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DCs were purified with the Pan Dendritic Cell Isolation Kit (Miltenyi Biotec) from spleens of WT mice injected with 1 × 106 B16-FLT3L cells. T cells were purified with the Naïve CD4+ T Cell Isolation Kit (Miltenyi Biotec) from the spleens and mLNs of pTreg TN/RKO mice and labeled with 2.5 μM CFSE (Sigma). Purity was always greater than 98% for both DCs and T cells. DCs (1 × 105) were incubated with sonicated bacterial extracts (25, 50, or 100 μg/ml) for 2 hours before the addition of 1 × 105 TN CD4+ T cells. Cell division was assessed by CFSE dilution and analyzed by flow cytometry after 3.5 days of coculture.
Bilate A.M., Bousbaine D., Mesin L., Agudelo M., Leube J., Kratzert A., Dougan S.K., Victora G.D, & Ploegh H.L. (2016). Tissue-specific emergence of regulatory and intraepithelial T cells from a clonal T cell precursor. Science immunology, 1(2), eaaf7471.
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9

Adoptive Transfer of IRF4-Overexpressing T Cells

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BALB/c splenic dendritic cells (DCs) were isolated by using the Pan
Dendritic Cell Isolation Kit (Miltenyi Biotec, San Diego, CA).
Irf4−/− CD4+ T cells
were activated for 3 days with BALB/c splenic DCs and 100 IU IL‐2
(PeproTech, Rocky Hill, NJ), and then transduced with IRF4‐GFP or
GFP‐Ctrl retrovirus as previously described.8 (link) Cells were cultured for 1 day after
transduction, and then adoptively transferred into Irf4fl/flCd4‐Cre mice on day 1 post‐heart
transplantation.
Zhang H., Wu J., Zou D., Xiao X., Yan H., Li X.C, & Chen W. (2018). Ablation of interferon regulatory factor 4 in T cells induces “memory” of transplant tolerance that is irreversible by immune checkpoint blockade. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 19(3), 884-893.
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10

Isolation of Dendritic Cells and Naive CD4+ T Cells

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DCs and total CD4+ T cells were isolated from spleens or colonic-LP of mice using the Pan Dendritic Cell Isolation Kit and CD4+ T Cells Isolation Kit (all from Miltenyi Biotec), respectively. Naive CD4+ T cells were isolated from spleens of mice using a Naive CD4+ T cell Isolation Kit (Miltenyi Biotec). Cell purity was assessed by flow cytometry and was consistently above 95%.
Duo L., Wu T., Ke Z., Hu L., Wang C., Teng G., Zhang W., Wang W., Ge Q., Yang Y, & Dai Y. (2020). Gain of Function of Ion Channel TRPV1 Exacerbates Experimental Colitis by Promoting Dendritic Cell Activation. Molecular Therapy. Nucleic Acids, 22, 924-936.
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