Blood mDCs and pDCs were enriched using the panDC enrichment kit (StemCell) and then sorted by FACS Aria (BD Biosciences) (purity >99.5%). Autologous total CD4+ T cells were purified using the EasySep Human CD4+ T Cell Enrichment Kit (StemCell). Allogeneic naïve CD4+ T cells (CD45RA+CD45RO−CCR7+) were enriched and FACS sorted. Culture medium consisted of RPMI 1640 (Gibco) supplemented with HEPES buffer, 2 mM L-glutamine, 1% nonessential amino acids, sodium pyruvate, 50 units/ml penicillin, 50 μg/ml streptomycin and 10% normal human serum AB (GemCell). L cells and OX40L-L cells were cultured in cRPMI containing 10% FCS and 600 ng/ml geneticin (Gibco). Monocyte-derived IL-4DCs and IFNDCs were generated as previously described (20 (link)).
Pan dc enrichment kit
Manufactured by STEMCELL
Sourced in Canada
The Pan-DC Enrichment Kit is a laboratory product designed to enrich for plasmacytoid dendritic cells (pDCs) from peripheral blood mononuclear cells (PBMCs). It utilizes a magnetic-based separation technique to isolate pDCs from the PBMC population.
Automatically generated - may contain errors
Lab products found in correlation
Xfe96 extracellular flux analyzer, by Agilent Technologies (2 mentions)
Easysep human cd4 t cell enrichment kit, by STEMCELL (1 mentions)
Geneticin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Facsaria, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsaria 2, by BD (1 mentions)
Moflow astrios, by Beckman Coulter (1 mentions)
Human cd14 isolation kit, by Miltenyi Biotec (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
5 protocols using pan dc enrichment kit
1
Isolation and Culture of Immune Cells
2
Isolation and Culture of Immune Cells
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All healthy (cancer-free) blood donors provided a written informed consent prior to inclusion in the study in accordance with the approval by the Institutional Review Boards at Baylor Research Institute. Mo-DCs were prepared by culturing purified blood monocytes from healthy individuals. Briefly, monocytes enriched from fresh peripheral blood mononuclear cells (PBMCs) or frozen elutriated cell fractions were cultured in DC culture medium (CellGenix) in the presence of 100 ng/ml human GM-CSF and 50 ng/ml IL-4 for 6 days. On day 3, culture medium was replaced with fresh medium containing the same concentrations of GM-CSF and IL-4. PBMCs of HLA-A*0201+ healthy donors were fractionated by elutriation. Total CD4+ and CD8+ T cells were enriched using enrichment kits (StemCell Technologies). Naïve CD8+ T cells (CD45RA+ CD45RO−) (purity > 99.2%) were further sorted on a FACSAria II (BD Biosciences). Monocytes and total B cells were purified using enrichment kits (StemCell Technologies). Blood myeloid DCs (mDCs, Lin-1− HLA-DR+ CD11c+ CD123−) and plasmacytoid DCs (pDCs, Lin-1− HLA-DR+ CD11c− CD123+) were pre-enriched using a pan-DC enrichment kit (StemCell Technologies) and then sorted. All flow cytometry data were collected on a FACSCanto II (BD Biosciences) and analyzed with FlowJo v9 (Tree Star).
3
Metabolic Profiling of Colonic DCs
4
Metabolic Profiling of Colonic DCs
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Colonic DCs were isolated from mice gavaged with P. UF or ΔlspA P. UF1 and orally infected with ΔactA L. m using a Pan-DC Enrichment Kit (StemCell Technologies, Vancouver, Canada). For real-time analysis of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), DCs were analyzed using an XFe96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA) as described.52 (link) Briefly, enriched colonic DCs (3 × 105 cells/well, pooled from 3 mice) were analyzed in non-buffered RPMI medium supplemented with 2.5 μM dextrose, 2 nM glutamine, and 1 μM sodium pyruvate. ECAR and OCR were analyzed in response to 1 μM oligomycin, 1.25 μM fluoro-carbonyl cyanide phenylhydrazone (FCCP), 1 μM rotenone and 1 μM antimycin A.
5
Isolation and Sorting of Human DC Subsets
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Whole blood and leukapheresis products from healthy volunteers were obtained for this study following approval from Mater Health Services Human Research Ethics Committee and with written informed consent. Peripheral blood mononuclear cells were isolated by Ficoll-Paque Plus density gradient centrifugation (GE Healthcare). For experiments comparing CD1c+ DC and CD14+ monocytes, CD1c+ DC were isolated to >85% purity using the human BDCA1 DC isolation kit and monocytes isolated to >98% purity using the human CD14 isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. For comparisons of human DC subsets, DC were first enriched using a pan DC enrichment kit (Stemcell Technologies), then labeled with fluorescently conjugated mouse anti-human Abs specific for CD3 (OKT3), CD14 (HCD14), CD16 (3G8), CD19 (HIB19), CD20 (2H7), CD56 (HCD56), CD1c (L161), CD141 (M80), CD123 (6H6), HLA-DR (L243), and Live/Dead Aqua Dye (all from Biolegend). Cells were sorted using a MoFlow® Astrios™ (Beckman Coulter) and human DC identified as live singlet cells that were lineage (CD3, CD14, CD16, CD19, CD20, CD56)− HLA-DR+ then further segregated by expression of CD141, CD1c, and CD123 (for pDC) (Figure S1 in Supplementary Material).
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