Colonic DCs were collected as described previously.34 (link) Briefly, the intestinal mucosa was washed in PBS to remove feces, placed in Hank’s balanced salt solution (HBSS) containing 5 mM EDTA, and incubated for 20 min with shaking. The tissue was cut into small pieces that were incubated in RPMI 1640 medium containing 4% fetal bovine serum (FBS), 1 mg/mL collagenase D (Roche Life Science, Mannheim, Germany), 0.5 mg/mL dispase (Invitrogen, Carlsbad, CA), and 50 μg/mL DNase I (Roche Life Science) for 50 min in a 37°C shaking water bath. Digested tissues were then resuspended in HBSS containing 5 mM EDTA and passed through a 40-μm cell strainer. Anti-CD11c MicroBeads (100 μL; Miltenyi Biotec, Bergisch Gladbach, Germany) were mixed with the cell pellet after it was resuspended in 500 μL buffer. The cell and microbead mixture was incubated for 15 min at 4°C, and then the cells were processed using the AutoMACS Pro separator (Miltenyi Biotec) according to the manufacturer’s protocol.
Anti cd11c microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD11c microbeads are magnetic beads conjugated with an antibody that recognizes the CD11c surface antigen. They are designed for the positive selection or depletion of CD11c-expressing cells from heterogeneous cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (4 mentions)
Ls column, by Miltenyi Biotec (4 mentions)
Collagenase d, by Roche (3 mentions)
Anti cd8 microbeads, by Miltenyi Biotec (3 mentions)
Gm csf, by Thermo Fisher Scientific (3 mentions)
Ms column, by Miltenyi Biotec (3 mentions)
Dnase 1, by Roche (2 mentions)
Automacs pro separator, by Miltenyi Biotec (2 mentions)
Anti cd11b microbead, by Miltenyi Biotec (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
L glutamine and penicillin streptomycin, by Merck Group (2 mentions)
Non essential amino acid solution, by Merck Group (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Recombinant murine gm csf, by Thermo Fisher Scientific (2 mentions)
Streptavidin microbeads, by Miltenyi Biotec (2 mentions)
Automacs, by Miltenyi Biotec (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
62 protocols using anti cd11c microbeads
1
Isolation of Colonic Dendritic Cells
2
Isolation and Purification of Tumor-associated Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Subcutaneous, orthotopic or MMTV-PyMT tumours were excised, cut in small pieces, treated with 10 U ml−1 collagenase I, 400 U ml−1 collagenase IV and 30 U ml−1 DNaseI (Worthington) for 30 min at 37 °C, squashed and filtered. Red blood cells were removed using erythrocyte lysis buffer and density gradients (Axis-Shield) were used to remove debris and dead cells.
Tumour-draining LNs were cut, dissociated with 10 U ml−1 collagenase I, 400 U ml−1 collagenase IV and 30 U mL−1 DNaseI (Worthington) for 45 min at 37 °C and filtered.
Spleens were flushed with 200 U ml−1 collagenase III (Worthington) and left for 30 min at 37 °C. Afterwards, spleens were filtered and red blood cells were removed using erythrocyte lysis buffer.
To purify DC subpopulations from tumour, spleen or LNs, CD11c+ cells were MACS-enriched (anti-CD11c microbeads; Miltenyi) and sorted using BD FACSAria II (BD Biosciences) according to the gating strategy inFig. 1a , Supplementary Fig. 6A or Supplementary Fig. 8A , respectively.
Bone marrow leukocytes were isolated through flushing of tibia and femur. The obtained cell suspensions were filtered, and red blood cells were removed using erythrocyte lysis buffer. To purify bone marrow monocytes, CD11b+ cells were MACS-enriched (anti-CD11b microbeads; Miltenyi) before sorting.
Tumour-draining LNs were cut, dissociated with 10 U ml−1 collagenase I, 400 U ml−1 collagenase IV and 30 U mL−1 DNaseI (Worthington) for 45 min at 37 °C and filtered.
Spleens were flushed with 200 U ml−1 collagenase III (Worthington) and left for 30 min at 37 °C. Afterwards, spleens were filtered and red blood cells were removed using erythrocyte lysis buffer.
To purify DC subpopulations from tumour, spleen or LNs, CD11c+ cells were MACS-enriched (anti-CD11c microbeads; Miltenyi) and sorted using BD FACSAria II (BD Biosciences) according to the gating strategy in
Bone marrow leukocytes were isolated through flushing of tibia and femur. The obtained cell suspensions were filtered, and red blood cells were removed using erythrocyte lysis buffer. To purify bone marrow monocytes, CD11b+ cells were MACS-enriched (anti-CD11b microbeads; Miltenyi) before sorting.
3
Isolation of CD8+ OT-I T Cells
4
Differentiation of Immune Cell Types
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human THP-1 cells (ATCC® TIB-202TM) were differentiated to macrophages (THP-1 macrophages) with 60 nM phorbol 12-myristate 13-acetate (PMA; Sigma) for 16 h, and cells were cultured for an additional 48 h without PMA. Human monocyte-derived dendritic cells (MDDC) were isolated by stimulating isolated monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF, 1000 U/ml) and IL-4 (250 U/ml) and maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FCS and 1% penicillin–streptomycin (Invitrogen-Gibco)35 (link),56 (link),57 (link). Bone marrow cells were isolated from the tibia and femur and cultured in RPMI1640 medium with 10% FBS, 1% penicillin–streptomycin, and 10% L929 conditioned media containing macrophage-colony stimulating factor (M-CSF) for 6 days, 25 ng/ml murine GM-CSF for 6–8 days to harvest BMDM or BMDC, respectively34 (link),35 (link). For IFN-α treatment, human primary plasmacytoid dendritic cells (pDC) and myeloid dendritic cells (mDC) were isolated35 (link),58 (link) and treated with IFN-α (100 U/ml) for 2 h. Mouse splenic CD11c + DCs were isolated from spleen of WT and PARP9 KO mice without or with VSV infection for 1 day using anti-CD11c microbeads (Miltenyi Biotec)34 (link).
5
Developing Th17 Cells with DCs and TLR Ligands
6
Generating M2-specific CD8 T cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
M282-specific memory CD8 T cells were induced using a DC-LM prime-boost immunization regimen as previously described.22 (link) Briefly, LPS-matured DCs were pulsed with M282–90 peptide and sorted via positive selection with anti-CD11c microbeads (Miltenyi Biotec, Auburn, CA). Naive BALB/c mice were primed with 5 × 105 DC-M282 intravenously (i.v.) and boosted 7 days later i.v. with 5 × 106 recombinant actA-deficient LM expressing the M282–90 epitope.
7
CD4+ Dendritic Cell RNA-Seq Analysis
8
CD4+ Dendritic Cell RNA-Seq Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleens were taken one hour after saline or SRBC immunization. CD4+ DCs were pre-enriched using MACS manual cell separation columns with anti-CD11c microbeads (Miltenyi Biotec) and further sorted based on surface markers of CD11c+I-Ab+CD4+CD8-. Cells were sorted twice on a FACSAira III to purities of over 99%. Sorted DCs (106) were snap frozen and then RNA was extracted with the QIAGEN RNeasy Kit. RNA quality was checked with the Agilent 2100 Bioanalyzer (RNA integrity number >9 for all samples). Barcoded sequencing libraries were generated with 100 ng of RNA with the Ovation RNA-Seq System V2 and Encore Rapid Library System. Sequencing was performed on an Illumina HiSeq 2500 (UCSF Human Genetics Core) with 100-bp paired-end reads. Sequences were reported as FASTQ files, which were aligned to the mm9 mouse genome with STAR (Spliced Transcript Alignment to a Reference). Generation of Log2FC values and further analyses were performed with a Bioconductor package on RStudio. The RNAseq data have been deposited in the Gene Expression Omnibus (NCBI) data repository under accession code GEO: GSE71165.
9
Dendritic Cell Priming and Boosting Strategies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DC priming and various boosting strategies were adapted from52 (link). Briefly, LPS-matured FMS-like tyrosine kinase-3 ligand (Flt-3L)-induced splenic DCs digested with collagenase/DNase were incubated for 2 h at 37 °C with 2 μM OVA(257–264) or GP33(33–41) peptide. CD11c+ cells were enriched using anti-CD11c Microbeads (Miltenyi Biotec) and ~5 × 105 peptide-coated DCs were intravenously (IV) injected into mice. Seven days later, mice were boosted IV with 1 × 107 CFU of recombinant attenuated (ActA−/−) Listeria monocytogenes expressing OVA (rLM-OVA) or GP33 (rLM-GP33), 2 × 106 PFU vaccinia virus expressing OVA (VACV-OVA), or 30 × 106 OVA(257–264) coated splenocytes. For boosting with peptide-coated splenocytes, single-cell suspensions of spleen cells from C57BL/6 mice were incubated with OVA(257–264) for 1 h at 37 °C in medium with fetal calf serum followed by several washes prior to determining cell number for cell transfer.
10
Investigating Prevotella-induced Cytokine Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow-derived dendritic cells (BMDCs) were obtained as previously described [30 (link)]. Briefly, bone marrow cells were isolated from 6- to 8-week-old C57BL/6 mice and cultured for 9 days with 20 ng/mL recombinant murine GM-CSF (PeproTech). Cells were purified by positive selection [26 (link)] using anti-CD11c microbeads (Miltenyi Biotec). The dendritic cell (DC) phenotype was controlled by flow cytometry. The cytokine response to Prevotella was assessed in 1 × 105 BMDCs incubated with LPS (10 ng/mL, Sigma) as a positive control, PBS as a negative control, or heat-killed (30 min at 60°C) Prevotella (1 × 107 cfu/well) for 24 h [15 (link)]. To compare the efficacy of Toll-like receptors (TLRs), the BMDC medium was supplemented with inhibitors of TLR2 [31 (link)] (C29, MCE, 50 μM) or TLR4 [32 (link)] (TAK-242, MCE, 100 nM). For all experiments, TLR inhibitors were added to the cells just prior to stimulation.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!