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Found 12 matching solutions for this experiment
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| Briefly, a single-cell suspension containing 1 × 108 PBMCs per mL of cell separation buffer (PBS, 3% FBS, 10mM EDTA) was prepared. |
Cells were then incubated for 10 min with 20 μL of MagniSort™ enrichment antibody cocktail per 100 μL of cell suspension. Cells were washed and resuspended in separation buffer. Then 10 μL of MagniSort™ negative selection beads were added per 100 μL of cell suspension. Following 5 min of incubation, a magnet was used to remove the bead-bound non-monocyte lymphocytes from the monocytes. |
. Enrichment efficiency was determined by flow cytometry staining with anti-human CD14 eFluor 450 (clone 613D, eBioscience) and anti-human CD16 APC (clone CB16, eBioscience). |
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| Briefly, a single-cell suspension containing 1 × 108 PBMCs per mL of cell separation buffer (PBS, 3% FBS, 10mM EDTA) was prepared. |
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| Cells were then incubated for 10 min with 20 μL of MagniSort™ enrichment antibody cocktail per 100 μL of cell suspension. Cells were washed and resuspended in separation buffer. Then 10 μL of MagniSort™ negative selection beads were added per 100 μL of cell suspension. Following 5 min of incubation, a magnet was used to remove the bead-bound non-monocyte lymphocytes from the monocytes. |
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| . Enrichment efficiency was determined by flow cytometry staining with anti-human CD14 eFluor 450 (clone 613D, eBioscience) and anti-human CD16 APC (clone CB16, eBioscience). |
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Human macrophages were obtained from peripheral blood mononuclear cells (PBMCs)
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Human macrophages were obtained from peripheral blood mononuclear cells (PBMCs)
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| Human monocytes were freshly isolated from peripheral blood of ALS patients and HC. |
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| Human monocytes were freshly isolated from peripheral blood of ALS patients and HC. |
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For negative selection, PBMCs were diluted to 5 × 107 cells/ml and incubated with 50 µl/ml isolation antibody cocktail and 50 µl/mL platelet removal cocktail for 5 min before the addition of 50 µl/ml RapidSpheres. After an additional 5 min of incubation, PBMCs were placed in the magnet for 3 min and the supernatant, containing monocytes, was collected and washed in PBS (1% FCS). |
The monocytes were resuspended in complete maturation medium and placed in the incubator. |
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| For negative selection, PBMCs were diluted to 5 × 107 cells/ml and incubated with 50 µl/ml isolation antibody cocktail and 50 µl/mL platelet removal cocktail for 5 min before the addition of 50 µl/ml RapidSpheres. After an additional 5 min of incubation, PBMCs were placed in the magnet for 3 min and the supernatant, containing monocytes, was collected and washed in PBS (1% FCS). |
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| The monocytes were resuspended in complete maturation medium and placed in the incubator. |
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| Subjects reported to the laboratory following an overnight fast and had forearm vein blood collected into a 10 ml K2EDTA vacutainer tube. |
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| Subjects reported to the laboratory following an overnight fast and had forearm vein blood collected into a 10 ml K2EDTA vacutainer tube. |
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Unwanted cells are labelled for removal with bispecific tetrameric antibody complexes recognizing the respective target antigen (CD2, CD3, CD16, CD19, CD20, CD56, CD66b, CD123, or glycophorin A) and dextran-coated magnetic particles. |
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| Unwanted cells are labelled for removal with bispecific tetrameric antibody complexes recognizing the respective target antigen (CD2, CD3, CD16, CD19, CD20, CD56, CD66b, CD123, or glycophorin A) and dextran-coated magnetic particles. |
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Enriched cells were labeled with CD3, CD19, CD20, CD56, CD66b, HLA-DR, CD14, and CD16 antibodies before sorting classical monocytes by FACS AriaII (BD). |
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| Enriched cells were labeled with CD3, CD19, CD20, CD56, CD66b, HLA-DR, CD14, and CD16 antibodies before sorting classical monocytes by FACS AriaII (BD). |
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| Human neutrophils and monocytes were isolated from venous blood from a healthy female donor. Peripheral blood mononuclear cells were obtained by gradient separation with Ficoll-Paque |
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| Human neutrophils and monocytes were isolated from venous blood from a healthy female donor. Peripheral blood mononuclear cells were obtained by gradient separation with Ficoll-Paque |
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Slan+ monocytes or CD14+ monocytes (2.5 × 104) were thereafter dispensed into U‐bottom 96‐well plates (Corning, Corning, NY, USA), and cultured for 24 h in RPMI 1640 medium containing 10% FBS, in the absence or in the presence of 20% cell‐free supernatants from SW480, SW620, and MCF7 cell lines. |
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| Slan+ monocytes or CD14+ monocytes (2.5 × 104) were thereafter dispensed into U‐bottom 96‐well plates (Corning, Corning, NY, USA), and cultured for 24 h in RPMI 1640 medium containing 10% FBS, in the absence or in the presence of 20% cell‐free supernatants from SW480, SW620, and MCF7 cell lines. |
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Macrophages were obtained from human peripheral blood monocytes. To obtain macrophages, monocytes were seeded on culture plastic and incubated for 7 days in DMEM/F12 medium with 2% fetal bovine serum. |
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| Macrophages were obtained from human peripheral blood monocytes. To obtain macrophages, monocytes were seeded on culture plastic and incubated for 7 days in DMEM/F12 medium with 2% fetal bovine serum. |
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Remaining cells were stained for FACS with anti-CD14, -16, -HLA-DR, and lineage exclusion markers as described for phenotyping above. |
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| Remaining cells were stained for FACS with anti-CD14, -16, -HLA-DR, and lineage exclusion markers as described for phenotyping above. |
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Purity was assessed for all cells isolated by positive and negative selection. |
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| Purity was assessed for all cells isolated by positive and negative selection. |
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