Anti cd11c macs beads, by Miltenyi Biotec - Product details

1

Detailed Dendritic Cell and Naive T Cell Sorting

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Cells were sorted using FACS Aria cell sorter flow cytometer (Becton Dickinson). For mesenteric dendritic cell sorting, cells were pre-enriched using anti-CD11c MACS beads (Miltenyi Biotec) and LS MACS Separation Columns (Miltenyi Biotec). Dendritic were sorted as Aqua⁻CD45⁺Lin⁻(CD3⁻B220⁻NK1.1⁻CD19⁻) CD11c^hi, and the subpopulations further as MHCII^intCD8α⁺CD11b^lo, MHCII^intCD8α⁻CD11b⁺, MHCII^hiCD103⁺CD11b⁻ and MHCII^hiCD103⁺CD11b⁺. Naïve CD4 T cells were pre-enriched by negative selection using biotinylated antibodies against CD8α, CD25, CD11c, CD11b, TER-119, NK1.1, and B220 and anti-biotin MACS beads (Miltenyi Biotec) and sorted as Aqua⁻CD11c⁻CD8α⁻MHCII⁻Vα2⁺CD4⁺CD25⁻CD62^hiCD44^lo.

Esterházy D., Loschko J., London M., Jove V., Oliveira T.Y, & Mucida D. (2016). Classical dendritic cells are required for dietary antigen-mediated peripheral regulatory T cell and tolerance induction. Nature immunology, 17(5), 545-555.

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2

Antigen-Specific CD8+ T Cell Activation

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DCs from pooled medLN or lung of day 12 X31-infected mice were purified on LS columns using anti-CD11c MACs beads (Miltenyi Biotec). CD8 T cells were isolated by MACs from the medLNs of day 7 X31-infected B6 mice or spleen from naive OT-I mice. All preparations were >95% pure as determined by flow cytometry. In some experiments, CD8 T cells were labeled for 10 min at 37°C with 5 µM CFSE (Molecular Probes). 2 × 10⁴ or 10⁵ DCs and 10⁵ CFSE-labeled CD8 T cells were cultured in 200 µl of complete medium in round-bottomed 96-well plates for 72 h at 37°C. In some experiments OVA protein, OVA peptide (OVA_257-264), or NP peptide (NP_366-374) were added to the cultures. Complete medium included RPMI 1640 supplemented with sodium pyruvate, Hepes, nonessential amino acids, penicillin, streptomycin, 2-mercaptoethanol, and 10% heat-inactivated FBS (Gibco). Memory CD8⁺CD44^hi T cells were sorted from donor mice using a FACSAria (BD) from preparations of splenocytes after positive selection with anti-CD8 MACS beads. An aliquot of sorted CD8⁺CD44^hi memory T cells was stained with flu NP MHC class I tetramers to calculate the number of NP⁺ T cells present in the sorted population. Equivalent numbers of CD8⁺CD44^hi NP⁺ T cells were transferred i.v. into 5 × 10³ naive CD45.1 recipient mice or 5 × 10⁴ μMT recipient mice.

León B., Ballesteros-Tato A., Randall T.D, & Lund F.E. (2014). Prolonged antigen presentation by immune complex–binding dendritic cells programs the proliferative capacity of memory CD8 T cells. The Journal of Experimental Medicine, 211(8), 1637-1655.

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3

Splenic DC Isolation and Gene Expression

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As previously described (Stanley et al., 2008 (link)) mice were infected with L. donovani and sacrificed 5 hours later. Spleens were digested in collagenase type IV (1 mg/ml; Worthington, Lakewood, NJ) and deoxyribonuclease I (0.5 mg/ml; Worthington) at RT for 45 minutes. Splenocytes were processed as described above. Splenic DCs were isolated from splenocyte preparations by positive selection with anti-CD11c MACS beads, according to the manufacturer’s instructions (Miltenyi Biotec). DCs were also stained for MHCII and CD86 and analyzed by flow cytometry, as described above. Purified DCs were stored in buffer RLT and homogenized in QIAshredder columns (both from QIAGEN). Total RNA was extracted from purified DC using the RNeasy Mini Kit with on-column DNase digestion (both from QIAGEN). RNA samples were reverse transcribed into cDNA using the cDNA Archive Kit (Applied Biosystems, Foster City, CA). RT-qPCR for Il12 and Il10 was performed on CF384 Touch Real-Time PRC Detection System (BIO-RAD) using the TaqMan Gene Expression Assay (Applied Bioscience). Relative quantification was performed using the comparative C_T method relative to Hprt and beta2m.

Kumar R., Bunn P.T., Singh S.S., Ng S.S., de Oca M.M., De Labastida Rivera F., Chauhan S.B., Singh N., Faleiro R.J., Edwards C.L., Frame T.C., Sheel M., Austin R.J., Lane S.W., Bald T., Smyth M.J., Hill G.R., Best S.E., Haque A., Corvino D., Waddell N., Koufariotis L., Mukhopadhay P., Rai M., Chakravarty J., Singh O.P., Sacks D., Nylen S., Uzonna J., Sundar S, & Engwerda C.R. (2020). Type I Interferons Suppress Anti-parasitic Immunity and Can Be Targeted to Improve Treatment of Visceral Leishmaniasis. Cell reports, 30(8), 2512-2525.e9.

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4

Detailed Dendritic Cell and Naive T Cell Sorting

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Cells were sorted using FACS Aria cell sorter flow cytometer (Becton Dickinson). For mesenteric dendritic cell sorting, cells were pre-enriched using anti-CD11c MACS beads (Miltenyi Biotec) and LS MACS Separation Columns (Miltenyi Biotec). Dendritic were sorted as Aqua⁻CD45⁺Lin⁻(CD3⁻B220⁻NK1.1⁻CD19⁻) CD11c^hi, and the subpopulations further as MHCII^intCD8α⁺CD11b^lo, MHCII^intCD8α⁻CD11b⁺, MHCII^hiCD103⁺CD11b⁻ and MHCII^hiCD103⁺CD11b⁺. Naïve CD4 T cells were pre-enriched by negative selection using biotinylated antibodies against CD8α, CD25, CD11c, CD11b, TER-119, NK1.1, and B220 and anti-biotin MACS beads (Miltenyi Biotec) and sorted as Aqua⁻CD11c⁻CD8α⁻MHCII⁻Vα2⁺CD4⁺CD25⁻CD62^hiCD44^lo.

Esterházy D., Loschko J., London M., Jove V., Oliveira T.Y, & Mucida D. (2016). Classical dendritic cells are required for dietary antigen-mediated peripheral regulatory T cell and tolerance induction. Nature immunology, 17(5), 545-555.

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5

Splenic DC Isolation and Gene Expression

Cited 1 time

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As previously described (Stanley et al., 2008 (link)) mice were infected with L. donovani and sacrificed 5 hours later. Spleens were digested in collagenase type IV (1 mg/ml; Worthington, Lakewood, NJ) and deoxyribonuclease I (0.5 mg/ml; Worthington) at RT for 45 minutes. Splenocytes were processed as described above. Splenic DCs were isolated from splenocyte preparations by positive selection with anti-CD11c MACS beads, according to the manufacturer’s instructions (Miltenyi Biotec). DCs were also stained for MHCII and CD86 and analyzed by flow cytometry, as described above. Purified DCs were stored in buffer RLT and homogenized in QIAshredder columns (both from QIAGEN). Total RNA was extracted from purified DC using the RNeasy Mini Kit with on-column DNase digestion (both from QIAGEN). RNA samples were reverse transcribed into cDNA using the cDNA Archive Kit (Applied Biosystems, Foster City, CA). RT-qPCR for Il12 and Il10 was performed on CF384 Touch Real-Time PRC Detection System (BIO-RAD) using the TaqMan Gene Expression Assay (Applied Bioscience). Relative quantification was performed using the comparative C_T method relative to Hprt and beta2m.

Kumar R., Bunn P.T., Singh S.S., Ng S.S., de Oca M.M., De Labastida Rivera F., Chauhan S.B., Singh N., Faleiro R.J., Edwards C.L., Frame T.C., Sheel M., Austin R.J., Lane S.W., Bald T., Smyth M.J., Hill G.R., Best S.E., Haque A., Corvino D., Waddell N., Koufariotis L., Mukhopadhay P., Rai M., Chakravarty J., Singh O.P., Sacks D., Nylen S., Uzonna J., Sundar S, & Engwerda C.R. (2020). Type I Interferons Suppress Anti-parasitic Immunity and Can Be Targeted to Improve Treatment of Visceral Leishmaniasis. Cell reports, 30(8), 2512-2525.e9.

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6

Isolation and Characterization of CD8+ OTI T Cells

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CD8⁺ OTI cells were purified from the spleens of naïve CD45.2⁺CD45.1⁻ B6.OTI or CD45.2⁺CD45.1⁺ B6.OTI mice by positive selection with the EasySep CD8⁺ positive selection kit (Stemcell Technologies). All T cell preparations were more than 95% pure as determined by flow cytometry. Where indicated, purified OTI cells were labeled with 1uL/mL CellTrace Violet (CTV) (Molecular Probes, ThermoFischer Scientific) for 20 minutes at 37°C. In the indicated experiments, purified OTI cells (6×10⁵) were transferred intravenously via the retro-orbital route into congenically different (B6.CD45.2⁻CD45.1⁺) recipient mice at the indicated time points. In the co-transfer experiments of LN-P and SP-P cells, secondary recipient mice received 4×10³ cells containing a 1:1 mixture of CD69⁺CD62^lo OTI cells sorted from the spleen (SP-P) and med-LN (LN-P) of previously PR8+OVA infected mice. DCs from pooled LNs and spleens were purified with LS columns using anti-CD11c MACs beads (Miltenyi). After staining with fluorochrome-conjugated antibodies, the indicated DC subsets were sorted. All sorting experiments were performed using a FACSAria (BD Biosciences) sorter in the University of Alabama at Birmingham Flow Cytometry core. All sorted DC and T cells populations were more than 95% pure as determined by flow cytometry.

Jenkins M.M., Bachus H., Botta D., Schultz M.D., Rosenberg A.F., León B, & Ballesteros-Tato A. (2021). Lung dendritic cells migrate to the spleen to prime long-lived TCF1hi memory CD8+ T cell precursors after influenza infection. Science immunology, 6(63), eabg6895.

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Murine Bone Marrow Dendritic and Macrophage Differentiation

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BMDC were generated from 6–8 week-old female C57BL/6 mice. Mice were maintained in the animal facility at the Lyman-Duff Medical Building (McGill University, Montreal, QC Canada) according to the McGill University Animal Care Committee (Permit #4543). This protocol respects the procedures on good animal practice provided by the Canadian Council for animal care. BMDC were obtained by differentiating precursor cells as previously described^{22 (link)}. Bone marrow cells (2.5 × 10⁵ cells/mL) were differentiated for 7 days in complete medium (RPMI 1640 supplemented with 5% heat-inactivated FBS, 10 mM HEPES, 2 mM L-glutamine 20 µg/mL gentamycin, and 50 µM β-ME) (Wisent, St-Bruno, QC, Canada) enriched with 20% Ag8.653-conditioned culture medium. CD11c⁺ BMDC were purified by positive selection using anti-CD11c MACS beads (Miltenyi Biotec, Auburn, CA). Purity was assessed routinely by flow cytometry, monitoring CD11c surface expression. BMDM were obtained by differentiating precursor murine bone marrow cells (5 × 10⁶) resuspended in culture medium (DMEM, 10% FBS, 2 mM L-glutamate, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µg/mL gentamicin, 2.5% HEPES, 55 µM β-ME, 1 mM sodium pyruvate) (Wisent) supplemented with 30% L929 fibroblast-conditioned culture medium for 7 days^{114 (link)}. Purity was assessed routinely by flow cytometry, monitoring CD11b and F4/80 co-expression.

Leroux L.P., Nasr M., Valanparambil R., Tam M., Rosa B.A., Siciliani E., Hill D.E., Zarlenga D.S., Jaramillo M., Weinstock J.V., Geary T.G., Stevenson M.M., Urban JF J.r., Mitreva M, & Jardim A. (2018). Analysis of the Trichuris suis excretory/secretory proteins as a function of life cycle stage and their immunomodulatory properties. Scientific Reports, 8, 15921.

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8

Infant vs. Adult mCD11b+ DCs RNA-seq

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mCD11b⁺DCs (B220⁻CD64⁻CD103⁻ CD11c⁺MHCII^hiCD11b⁺) from adult (7wks-old) and infant (2-3wks-old) mice were sorted from the mLN 24h after HDM+LPS sensitization using a FACSAria (BD Biosciences) after positive selection with anti-CD11c MACS beads (Miltenyi Biotec). RNA was isolated from the sorted cells using the RNeasy Plus Micro RNA purification kit (QIAGEN). Three or four replicates from three or four independent experiments for each condition were analyzed with RNA-seq. Library preparation and RNA sequencing was conducted through Genewiz. Libraries were sequenced using a 1Å~ 50-bp single end rapid run on the HiSeq2500 platform. Sequence reads were trimmed to remove possible adaptor sequences and nucleotides with poor quality (error rate < 0.05) at the end. After trimming, sequence reads shorter than 30 nucleotides were discarded. Remaining sequence reads were mapped to the Mus musculus mm10 reference genome using CLC genomics workbench v. 9.0.1. Differential gene expression was determined using DESeq2. Genes with an adjusted P value < 0.05 and an absolute log1.5-fold change (ratio +/−0.58) were considered significantly differentially expressed genes between adult and infant mCD11b⁺DCs.

Bachus H., Kaur K., Papillion A.M., Marquez-Lago T.T., Yu Z., Ballesteros-Tato A., Matalon S, & León B. (2019). Impaired Tumor Necrosis Factor-α-driven Dendritic Cell Activation Limits Lipopolysaccharide-induced Protection from Allergic Inflammation in Infants.. Immunity, 50(1), 225-240.e4.

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Anti cd11c macs beads

Lab products found in correlation

8 protocols using anti cd11c macs beads

Detailed Dendritic Cell and Naive T Cell Sorting

Antigen-Specific CD8+ T Cell Activation

Splenic DC Isolation and Gene Expression

Detailed Dendritic Cell and Naive T Cell Sorting

Splenic DC Isolation and Gene Expression

Isolation and Characterization of CD8+ OTI T Cells

Murine Bone Marrow Dendritic and Macrophage Differentiation

Infant vs. Adult mCD11b+ DCs RNA-seq

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