DCs were isolated using RosetteSep human monocyte enrichment cocktail (StemCell Technologies) according to the manufacturer’s instructions. In brief, blood from buffy coats from healthy donors was incubated for 20 min with RosetteSep human monocyte enrichment cocktail (StemCell Technologies), layered on top of Ficoll-Paque Plus (GE HealthCare), and centrifuged at 1,200 × g for 20 min without acceleration or braking. The monocyte-containing layer was recovered, and cells were washed with PBS and passed through a 100-μm-pore-size cell strainer. Monocytes were then differentiated for 6 days in RPMI medium containing 10% FBS supplemented with 37.5 ng/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) (PeproTech) and 37.5 ng/ml of interleukin-4 (IL-4) (PeproTech), with changing of the medium after 4 days. For experiments, cells were resuspended in RPMI medium containing 10% FBS.
Rosettesep human monocyte enrichment cocktail
Manufactured by STEMCELL
Sourced in Canada, United States, France
The RosetteSep Human Monocyte Enrichment Cocktail is a cell separation product designed to isolate monocytes from human peripheral blood mononuclear cells. It uses an immunodensity-based separation technique to enrich for monocytes.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (9 mentions)
L glutamine, by Thermo Fisher Scientific (8 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (6 mentions)
Lymphoprep, by STEMCELL (4 mentions)
Sepmate 50 ml tubes, by STEMCELL (4 mentions)
Ficoll paque, by GE Healthcare (3 mentions)
Penstrep, by Roche (3 mentions)
Ovcar3, by American Type Culture Collection (3 mentions)
Mcdb105 medium199, by Corning (3 mentions)
Aim 5 medium, by Thermo Fisher Scientific (3 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Human serum, by Merck Group (2 mentions)
Rhgm csf, by Thermo Fisher Scientific (2 mentions)
Rhil 4, by R&D Systems (2 mentions)
M csf, by R&D Systems (2 mentions)
Green cmfda, by Thermo Fisher Scientific (2 mentions)
Balb c rag2 γc mice, by Thermo Fisher Scientific (2 mentions)
Immunocult sf macrophage medium, by STEMCELL (2 mentions)
Ix penstrep, by Roche (2 mentions)
Anti cd3 apc cy7, by BioLegend (2 mentions)
79 protocols using rosettesep human monocyte enrichment cocktail
1
Isolation and Differentiation of Human Dendritic Cells
2
Monocyte Activation and NF-κB Inhibition
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Monocytes were enriched from the peripheral blood samples of healthy individuals by negative selection using the RosetteSep™ Human Monocyte Enrichment Cocktail (StemCell Technologies, Vancouver, Canada) according to the manufacturer's instructions. The purity of monocytes was ≥90%. Approximately 1 × 105 monocytes were pretreated with different doses (0, 1, 5, and 25 μM) of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB activation [36 (link), 37 (link)]. Monocytes were then stimulated for 6 h, under adherent conditions, with CMPs or 5 μg/mL Lipopolysaccharide (LPS from Escherichia coli, serotype O111:B4; InvivoGen, San Diego, CA) (positive control). All cultures were performed at 37 °C and 5% CO2 in 96-well flat-bottom plates in a final volume of 200 μL of RPMI-1640 supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% iFBS previously filtered through a 0.20 μm-Acrodiscs® syringe filter. After incubation, the cell surface expression of activation markers (HLA-DR and CD69) was evaluated by flow cytometry.
The viability of PDTC-treated cells was assessed with propidium iodide (PI) and DIOC6 staining. Additionally, to rule out any effect of 25 μM PDTC on the cellular ability to bind and uptake MPs, PBMCs treated or not with PDTC were incubated with CFSE-labeled CMPs for 1 h to evaluate the percentages of binding and uptake of MPs by cells.
The viability of PDTC-treated cells was assessed with propidium iodide (PI) and DIOC6 staining. Additionally, to rule out any effect of 25 μM PDTC on the cellular ability to bind and uptake MPs, PBMCs treated or not with PDTC were incubated with CFSE-labeled CMPs for 1 h to evaluate the percentages of binding and uptake of MPs by cells.
3
Isolation of Myeloid APCs from Whole Blood
4
Isolation and Differentiation of Primary Immune Cells
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Human primary T cells and monocytes were isolated from healthy subjects’ peripheral blood by using RosetteSep human T cell enrichment cocktail and RosetteSep human monocyte enrichment cocktail (Stemcell technologies, Vancouver, BC, Canada), followed by Ficoll Paque gradient centrifugation. Macrophages were further derived from monocytes via replacing half of the culture medium each day for 7 days.
5
Cell Sorting and RNA Extraction
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For cell sorting, blood cells were enriched using the RosetteSep Human Monocyte Enrichment Cocktail (StemCell Technologies, Vancouver, BC, Canada). Blood and BAL cells were used within 1 h after retrieval from the study subjects and stained with a validated panel of antibodies (supplementary table S1 ). For cell sorting, FACS Aria Fusion or AriaIII (both BD) were used. Sorted cells were subsequently resuspended in Qiazol Lysis reagent (Qiagen, Venlo, the Netherlands) and stored at −80°C until further use.
6
Isolation and Enrichment of Human Monocytes
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PBMCs were isolated using density gradient centrifugation by layering blood onto Biocoll (Millipore) and centrifugation at 1200×g for 20 min without brake. The resulting buffy layer was then washed twice with PBS before use in subsequent experiments. Monocyte enrichment was performed using the RosetteSep™ Human Monocyte Enrichment Cocktail (Stemcell Technologies). Positive enrichment of CD14+ monocytes was performed using CD14 Magnetic Particles (Becton–Dickinson). Cells were counted using trypan blue staining on a Neubauer hemocytometer and cultured at 37 °C with 5 % CO2, in RPMI 1640 supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM l -glutamine (all Biochrom), and 10 % AB serum.
7
Generating Dendritic Cells from Monocytes
8
Isolation of Immune Cells from Blood
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Neutrophils were isolated from fresh venous blood collected in lithium heparin vacutainers (BD, Scoresby, Australia) using the EasySep Direct Human Neutrophil Isolation Kit (STEMCELL Technologies, Tullamarine, Australia) as per manufacturer’s instructions. Neutrophil purity was assessed by cell morphology using light microscopy of Giemsa stained smears of the isolated cells, and viability was assessed using trypan blue exclusion. Monocytes were isolated from both fresh venous blood, collected in lithium heparin vacutainers (BD), as well as from buffy coats supplied from the Australian Red Cross Blood Service. Monocytes were isolated by negative selection using the RosetteSep Human Monocyte Enrichment Cocktail (STEMCELL Technologies) as per manufacturer’s instructions. Monocyte purity was assessed by staining (anti-CD14 antibody, BioLegend, San Diego, CA) and measuring CD14+ cells by flow cytometry. Monocytes were either frozen in fetal bovine serum (FBS) in 20% dimethyl sulfoxide (DMSO) in liquid nitrogen for later use or used immediately after isolation. Natural killer (NK) cells were isolated from fresh venous blood collected in sodium heparin vacutainers (BD). NK cells were isolated by negative selection using the RosetteSep Human NK Enrichment Cocktail (STEMCELL Technologies) as per manufacturer’s instructions.
9
Isolation and Culture of Primary Human Monocytes
10
Cell Enrichment and Labeling Protocol
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PBMCs were enriched for CD14+ cells using RosetteSep™ Human Monocyte Enrichment Cocktail (STEMCELL, Manchester, Greater Manchester, UK). PBMC or umbilical cord blood cells were enriched for CD34+ or CD3+ cells or depleted of CD3+, CD4+, CD8+ or CD25+ cells by the magnetic cell sorting system (MACS) (Miltenyi Biotec, Bisley, Surrey, UK). PBMC or T cells were labelled with CFSE as required (Sigma-Aldrich Ltd, Gillingham, Dorset, UK). All procedures were performed according to the manufacturer's instructions.
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