Isolation:Article Title: The human cytomegalovirus-encoded pUS28 antagonizes CD4+ T cell recognition by targeting CIITA
Article Snippet: .. DOI: https://doi.org/10.7554/eLife.96414 18 of 28 Generation of monocyte-derived dendritic cells To obtain human dendritic cells, monocytes were isolated from PBMCs using a CD14 MicroBead kit (Miltenyi) according to the manufacturer’s instructions, and purity was validated by flow cytometry. .. For maturation of monocytes into dendritic cells, monocytes were first cultured for 3 d in RPMI 1640 ( c. c. pro) supplemented with 10% (v/v) heat- inactivated human serum (Merck), 50 U/ml IL- 2 (Miltenyi), 10 ng/ml IL- 7, 5 ng/ml IL- 15 (R&D Systems), 10 ng/ml IL- 4 (R&D Systems) and 100 ng/ml GMCSF (Miltenyi).
Article Title: The human cytomegalovirus-encoded pUS28 antagonizes CD4+ T cell recognition by targeting CIITA
Article Snippet: .. To obtain human dendritic cells, monocytes were isolated from PBMCs using a CD14 MicroBead kit (Miltenyi) according to the manufacturer’s instructions, and purity was validated by flow cytometry. .. For maturation of monocytes into dendritic cells, monocytes were first cultured for 3 d in RPMI 1640 (c.c.pro) supplemented with 10% (v/v) heat-inactivated human serum (Merck), 50 U/ml IL-2 (Miltenyi), 10 ng/ml IL-7, 5 ng/ml IL-15 (R&D Systems), 10 ng/ml IL-4 (R&D Systems) and 100 ng/ml GMCSF (Miltenyi).
Article Title: Compounds and methods targeting epiregulin
Article Snippet: Briefly, PBMCs are isolated using density-gradient centrifugation with Ficoll (#17-1440-02, GE Healthcare) and Sepmate 50 (#15450, STEMCELL Technologies) from LRS-WBC. .. CD14+ monocytes are isolated using positive selection with a CD14+ microbead kit (#130-050-201, Miltenyi Biotec) following the manufacturer's manual. .. Cells are then cultured at 1 million/ml with 1000 unit/ml GM-CSF and 600 unit/ml IL-4 for 6 days to drive to immature dendritic cells (MDDC) in RPMI medium with L-glutamine and 25 mM HEPES supplemented with 10% FBS, 1 mM sodium pyruvate, 1× penicillin-streptomycin, 1× non-essential amino acids, and 55 μM 2-mercaptoethanol (hereafter referred to complete RPMI medium or medium, purchased from Life Technologies).
Article Title: Monocyte-macrophage membrane expression of IL-1R2 is a severity biomarker in sepsis
Article Snippet: Data were analyzed with FlowJo software (Treestar, Ashland, US). .. Monocytes were isolated from buffy coats from healthy donors using the CD14 MicroBead Kit (Miltenyi Biotec). .. The cells were primed for 4 days with 50 ng/mL of recombinant human (rh) Macrophage Colony-Stimulating Factor (M-CSF) or rh Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) (Peprotech) in RPMI 1640 (Lonza) with 10% FBS (Lonza), 1% l -glutamine (Lonza) and 1% Penicillin-Streptomycin (Lonza).
Article Title: Monocyte-macrophage membrane expression of IL-1R2 is a severity biomarker in sepsis.
Article Snippet: Data were analyzed with FlowJo software (Treestar, Ashland, US). .. Monocytes were isolated from buffy coats from healthy donors using the CD14 MicroBead Kit (Miltenyi Biotec). .. The cells were primed for 4 days with 50 ng/mL of recombinant human (rh) Macrophage ColonyStimulating Factor (M-CSF) or rh Granulocyte-Macrophage ColonyStimulating Factor (GM-CSF) (Peprotech) in RPMI 1640 (Lonza) with 10% FBS (Lonza), 1% L-glutamine (Lonza) and 1% Penicillin-Streptomycin (Lonza).
Article Title: Circulating pre-osteoclasts are primed for osteoclast fate and synovial tissue homing in psoriatic arthritis
Article Snippet: .. After PBMC isolation as described above, monocyte enrichment was carried out using positive selection with the CD14 MicroBead kit (Miltenyi, 130-050-201) as per the manufacturer’s instructions. .. Cells were blocked with FACS buffer at 80μL per 10 million cells, and labelled with CD14 MicroBeads at 20μL per 10 million cells for 15minutes at 4°C.
Flow Cytometry:Article Title: The human cytomegalovirus-encoded pUS28 antagonizes CD4+ T cell recognition by targeting CIITA
Article Snippet: .. DOI: https://doi.org/10.7554/eLife.96414 18 of 28 Generation of monocyte-derived dendritic cells To obtain human dendritic cells, monocytes were isolated from PBMCs using a CD14 MicroBead kit (Miltenyi) according to the manufacturer’s instructions, and purity was validated by flow cytometry. .. For maturation of monocytes into dendritic cells, monocytes were first cultured for 3 d in RPMI 1640 ( c. c. pro) supplemented with 10% (v/v) heat- inactivated human serum (Merck), 50 U/ml IL- 2 (Miltenyi), 10 ng/ml IL- 7, 5 ng/ml IL- 15 (R&D Systems), 10 ng/ml IL- 4 (R&D Systems) and 100 ng/ml GMCSF (Miltenyi).
Article Title: The human cytomegalovirus-encoded pUS28 antagonizes CD4+ T cell recognition by targeting CIITA
Article Snippet: .. To obtain human dendritic cells, monocytes were isolated from PBMCs using a CD14 MicroBead kit (Miltenyi) according to the manufacturer’s instructions, and purity was validated by flow cytometry. .. For maturation of monocytes into dendritic cells, monocytes were first cultured for 3 d in RPMI 1640 (c.c.pro) supplemented with 10% (v/v) heat-inactivated human serum (Merck), 50 U/ml IL-2 (Miltenyi), 10 ng/ml IL-7, 5 ng/ml IL-15 (R&D Systems), 10 ng/ml IL-4 (R&D Systems) and 100 ng/ml GMCSF (Miltenyi).
Selection:Article Title: Compounds and methods targeting epiregulin
Article Snippet: Briefly, PBMCs are isolated using density-gradient centrifugation with Ficoll (#17-1440-02, GE Healthcare) and Sepmate 50 (#15450, STEMCELL Technologies) from LRS-WBC. .. CD14+ monocytes are isolated using positive selection with a CD14+ microbead kit (#130-050-201, Miltenyi Biotec) following the manufacturer's manual. .. Cells are then cultured at 1 million/ml with 1000 unit/ml GM-CSF and 600 unit/ml IL-4 for 6 days to drive to immature dendritic cells (MDDC) in RPMI medium with L-glutamine and 25 mM HEPES supplemented with 10% FBS, 1 mM sodium pyruvate, 1× penicillin-streptomycin, 1× non-essential amino acids, and 55 μM 2-mercaptoethanol (hereafter referred to complete RPMI medium or medium, purchased from Life Technologies).
Article Title: Circulating pre-osteoclasts are primed for osteoclast fate and synovial tissue homing in psoriatic arthritis
Article Snippet: .. After PBMC isolation as described above, monocyte enrichment was carried out using positive selection with the CD14 MicroBead kit (Miltenyi, 130-050-201) as per the manufacturer’s instructions. .. Cells were blocked with FACS buffer at 80μL per 10 million cells, and labelled with CD14 MicroBeads at 20μL per 10 million cells for 15minutes at 4°C.
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