other:
Flow Cytometry:Article Title: IL-7–dependent and –independent lineages of IL-7R–dependent human T cells
Article Snippet: Subsets were manually gated with FlowJo v10 (FlowJo, LLC) and further analyzed in R. The cellular composition was visualized with the data downsampled to 50,000 cells per sample through Uniform Manifold Approximation and Projection (UMAP) based on the expression levels of the following markers: CD3, CD4, CD8, CD11c, CD14, CD16, CD19, CD20, CD56, CD117, CD123, CD127, CD161, CRTH2, HLA-DR, γδTCR, Vδ1, Vδ2, iNKT, and MR1. .. Flow cytometry for NK cell and ILC subsets Flow cytometry analysis was performed on PBMCs labeled with the following antibodies: biotinylated anti-human antibodies against CD1a (REA736; Miltenyi Biotec), CD14 (REA599; Miltenyi Biotec), CD66abce (REA1230; Miltenyi Biotec), CD123 (REA918; Miltenyi Biotec), CD203c (REA826; Miltenyi Biotec), CD303 (REA693; Miltenyi Biotec) and FceRIa (AER37(CRA-1); BioLegend), together with streptavidin BV570 or PE-Cy5 (BioLegend); and conjugated anti-human CD45 Alexa Fluor 532 (HI30; Thermo Fisher Scientific, CD34 Red 718 (581, BD Biosciences), CD3 BB790-P (UCHT1; BD Biosciences), CD8b BV480 (2ST8.5H7; BD Biosciences), CD4 NovaFluor Blue 660-120S (SK3; Thermo Fisher Scientific), CD5 PerCP (UCHT2; BioLegend) or BUV615 (UCHT2; BD Biosciences) CD19 BV570 or PE-Cy5 (HIB19; BioLegend), CD7 BUV496 or BV510 (M-T701; BD Biosciences), CD94 BUV661 or BV750 (HP-3D9; BD Biosciences), CD16 PE-Alexa Fluor 700 (3G8; Thermo Fisher Scientific), CD56 BB755-P or BUV661 (NCAM16.2; BD Biosciences), NKG2A APC-Fire 750 (S19004C; BioLegend), NKG2C PE-Vio615 (REA205; Miletnyi Biotec), CD57 BV785 (QA17A04; BioLegend), CD127 PE-Cy7 (eBioRDR5; Thermo Fisher Scientific), CD161 BUV395 (HP-3G10; BD Biosciences), CD25 BV750 or BUV563 (2A3; BD Biosciences), CD117 BV605 (104D2; BioLegend), CRTH2 Alexa Fluor 647 (BM16; BD Biosciences), CD45RA BUV496 (HI100; BD Biosciences), NKp44 BB630-P2 (p44-8; BD Biosciences) and EOMES (WD1928; Thermo Fisher Scientific) Abs. .. Fc receptors were blocked with IgG from human serum (Millipore Sigma) and non-specific dye binding was inhibited with CellBlox blocking buffer (Thermo Fisher Scientific).
Article Title: CD116 + fetal precursors migrate to the perinatal lung and give rise to human alveolar macrophages
Article Snippet: The indicated cell populations were sorted into RPMI 1640/30% FCS medium using a BD FACSAria III (BD Biosciences) or a MA900 (Sony Biotechnology) cell sorter. .. The following antibodies were used for flow cytometry and cell sorting: biotinylated anti-human CD3 (OKT3; Thermo Fisher Scientific or UCHT1; BioLegend), CD19 (HIB19; Thermo Fisher Scientific or BioLegend), CD20 (2H7; BioLegend), CD56 (HCD56; BioLegend), CD66abce (REA1230 or TET2; Miltenyi Biotec), CD94 (REA113; Miltenyi Biotec), NKp46 (9E2; BioLegend), TCRαβ (IP26; BioLegend), TCRγδ (B1; BD Biosciences), HLA-B12 (REA138; Miltenyi Biotec); conjugated anti-human CD1c BV785 (L161; BioLegend), CD1c BV650 (L161; BioLegend), CD11b PerCP-Cy5.5 (ICRF44; BioLegend), CD11b BUV737 (M1/70; BD Biosciences), CD11b BB515 (ICRF44; BD Biosciences), CD11c R718 (B-ly6; BD Biosciences), CD14 BV421 (M5E2; BioLegend), CD14 BV480 (MφP9; BD Biosciences), CD16 PE-Cy7 (3G8; BioLegend), CD33 BUV805 (WM53; BD Biosciences), CD34 PE-Dazzle594 (581; BioLegend), CD34 BV650 (563; BD Biosciences), CD34 FITC (581; BD Biosciences), CD45 APC-Cy7 (HI30; BioLegend), CD45 BUV395 (HI30; BD Biosciences), CD45 BV785 (HI30; BioLegend), CD64 BUV737 (10.1; BD Biosciences), CD64 PE-Dazzle594 (10.1; BioLegend), CD88 APC (S5/1; BioLegend), CD88 PE-Cy7 (S5/1; BioLegend), CD115 PE-Cy7 (9-4D2-1E4; BioLegend), CD116 PE (4H1; Thermo Fisher Scientific or BioLegend), CD141 BV785 (M80; BioLegend), CD141 BV650 (1A4; BD Biosciences), CD163 BV650 (GHI/61; BD Biosciences), CD169 APC (7–239; BioLegend), CD169 BV421 (7–239; BioLegend), CD206 BV605 (19.2; BD Biosciences), CX3CR1 BB515 (2A9-1; BD Biosciences), HLA-A2 AF700 (BB7.2; BioLegend), HLA-A9 APC (REA127; Miltenyi Biotec), HLA-B8 APC-Cy7 (REA145; Miltenyi Biotec), HLA-DR BUV395 (G46-6; BD Biosciences), HLA-DR APC-Fire750 (L243; BioLegend), HLA-DR BV650 (L243; BioLegend), and conjugated anti-mouse CD45 AF700 (30-F11; BioLegend). ..
Article Title: Human influenza virus infection elicits distinct patterns of monocyte and dendritic cell mobilization in blood and the nasopharynx
Article Snippet: Excess PBMCs were cryopreserved in FBS (Gibco) + 10% DMSO (Sigma) and stored in liquid nitrogen. .. Phenotypic analysis was performed on NPA cells and PBMCs using Live/Dead Blue; lineage markers CD3 (SK7; BD), CD19 (HIB19; Biolegend), CD20 (L27, BD), CD45 (HI30; BD), CD56 (HCD56, BD) and CD66abce (TET2, Miltenyi Biotec); HLA-DR (TU36, Life Technologies), CD14 (M5E2, BD), CD16 (3GE, Biolegend), CD11c (B-Ly6, BD), CD1c (AD5-8E7; Miltenyi), CD141 (AD5-14H12; Miltenyi), CD123 (7G3; BD); maturation markers CD83 (HB15e, Biolegend) and CD86 (2331; BD); adhesion marker CD62L (SK11, BD); and migration markers CCR2 (K036C2, Biolegend) and CCR7 (150503: BD); and fixed with 1% paraformaldehyde for flow cytometry. .. Samples were acquired on an LSRFortessa flow cytometer (BD Biosciences) and analysed using FlowJo software v10 (Tree Star).
Labeling:Article Title: IL-7–dependent and –independent lineages of IL-7R–dependent human T cells
Article Snippet: Subsets were manually gated with FlowJo v10 (FlowJo, LLC) and further analyzed in R. The cellular composition was visualized with the data downsampled to 50,000 cells per sample through Uniform Manifold Approximation and Projection (UMAP) based on the expression levels of the following markers: CD3, CD4, CD8, CD11c, CD14, CD16, CD19, CD20, CD56, CD117, CD123, CD127, CD161, CRTH2, HLA-DR, γδTCR, Vδ1, Vδ2, iNKT, and MR1. .. Flow cytometry for NK cell and ILC subsets Flow cytometry analysis was performed on PBMCs labeled with the following antibodies: biotinylated anti-human antibodies against CD1a (REA736; Miltenyi Biotec), CD14 (REA599; Miltenyi Biotec), CD66abce (REA1230; Miltenyi Biotec), CD123 (REA918; Miltenyi Biotec), CD203c (REA826; Miltenyi Biotec), CD303 (REA693; Miltenyi Biotec) and FceRIa (AER37(CRA-1); BioLegend), together with streptavidin BV570 or PE-Cy5 (BioLegend); and conjugated anti-human CD45 Alexa Fluor 532 (HI30; Thermo Fisher Scientific, CD34 Red 718 (581, BD Biosciences), CD3 BB790-P (UCHT1; BD Biosciences), CD8b BV480 (2ST8.5H7; BD Biosciences), CD4 NovaFluor Blue 660-120S (SK3; Thermo Fisher Scientific), CD5 PerCP (UCHT2; BioLegend) or BUV615 (UCHT2; BD Biosciences) CD19 BV570 or PE-Cy5 (HIB19; BioLegend), CD7 BUV496 or BV510 (M-T701; BD Biosciences), CD94 BUV661 or BV750 (HP-3D9; BD Biosciences), CD16 PE-Alexa Fluor 700 (3G8; Thermo Fisher Scientific), CD56 BB755-P or BUV661 (NCAM16.2; BD Biosciences), NKG2A APC-Fire 750 (S19004C; BioLegend), NKG2C PE-Vio615 (REA205; Miletnyi Biotec), CD57 BV785 (QA17A04; BioLegend), CD127 PE-Cy7 (eBioRDR5; Thermo Fisher Scientific), CD161 BUV395 (HP-3G10; BD Biosciences), CD25 BV750 or BUV563 (2A3; BD Biosciences), CD117 BV605 (104D2; BioLegend), CRTH2 Alexa Fluor 647 (BM16; BD Biosciences), CD45RA BUV496 (HI100; BD Biosciences), NKp44 BB630-P2 (p44-8; BD Biosciences) and EOMES (WD1928; Thermo Fisher Scientific) Abs. .. Fc receptors were blocked with IgG from human serum (Millipore Sigma) and non-specific dye binding was inhibited with CellBlox blocking buffer (Thermo Fisher Scientific).
FACS:Article Title: CD116 + fetal precursors migrate to the perinatal lung and give rise to human alveolar macrophages
Article Snippet: The indicated cell populations were sorted into RPMI 1640/30% FCS medium using a BD FACSAria III (BD Biosciences) or a MA900 (Sony Biotechnology) cell sorter. .. The following antibodies were used for flow cytometry and cell sorting: biotinylated anti-human CD3 (OKT3; Thermo Fisher Scientific or UCHT1; BioLegend), CD19 (HIB19; Thermo Fisher Scientific or BioLegend), CD20 (2H7; BioLegend), CD56 (HCD56; BioLegend), CD66abce (REA1230 or TET2; Miltenyi Biotec), CD94 (REA113; Miltenyi Biotec), NKp46 (9E2; BioLegend), TCRαβ (IP26; BioLegend), TCRγδ (B1; BD Biosciences), HLA-B12 (REA138; Miltenyi Biotec); conjugated anti-human CD1c BV785 (L161; BioLegend), CD1c BV650 (L161; BioLegend), CD11b PerCP-Cy5.5 (ICRF44; BioLegend), CD11b BUV737 (M1/70; BD Biosciences), CD11b BB515 (ICRF44; BD Biosciences), CD11c R718 (B-ly6; BD Biosciences), CD14 BV421 (M5E2; BioLegend), CD14 BV480 (MφP9; BD Biosciences), CD16 PE-Cy7 (3G8; BioLegend), CD33 BUV805 (WM53; BD Biosciences), CD34 PE-Dazzle594 (581; BioLegend), CD34 BV650 (563; BD Biosciences), CD34 FITC (581; BD Biosciences), CD45 APC-Cy7 (HI30; BioLegend), CD45 BUV395 (HI30; BD Biosciences), CD45 BV785 (HI30; BioLegend), CD64 BUV737 (10.1; BD Biosciences), CD64 PE-Dazzle594 (10.1; BioLegend), CD88 APC (S5/1; BioLegend), CD88 PE-Cy7 (S5/1; BioLegend), CD115 PE-Cy7 (9-4D2-1E4; BioLegend), CD116 PE (4H1; Thermo Fisher Scientific or BioLegend), CD141 BV785 (M80; BioLegend), CD141 BV650 (1A4; BD Biosciences), CD163 BV650 (GHI/61; BD Biosciences), CD169 APC (7–239; BioLegend), CD169 BV421 (7–239; BioLegend), CD206 BV605 (19.2; BD Biosciences), CX3CR1 BB515 (2A9-1; BD Biosciences), HLA-A2 AF700 (BB7.2; BioLegend), HLA-A9 APC (REA127; Miltenyi Biotec), HLA-B8 APC-Cy7 (REA145; Miltenyi Biotec), HLA-DR BUV395 (G46-6; BD Biosciences), HLA-DR APC-Fire750 (L243; BioLegend), HLA-DR BV650 (L243; BioLegend), and conjugated anti-mouse CD45 AF700 (30-F11; BioLegend). ..
Marker:Article Title: Human influenza virus infection elicits distinct patterns of monocyte and dendritic cell mobilization in blood and the nasopharynx
Article Snippet: Excess PBMCs were cryopreserved in FBS (Gibco) + 10% DMSO (Sigma) and stored in liquid nitrogen. .. Phenotypic analysis was performed on NPA cells and PBMCs using Live/Dead Blue; lineage markers CD3 (SK7; BD), CD19 (HIB19; Biolegend), CD20 (L27, BD), CD45 (HI30; BD), CD56 (HCD56, BD) and CD66abce (TET2, Miltenyi Biotec); HLA-DR (TU36, Life Technologies), CD14 (M5E2, BD), CD16 (3GE, Biolegend), CD11c (B-Ly6, BD), CD1c (AD5-8E7; Miltenyi), CD141 (AD5-14H12; Miltenyi), CD123 (7G3; BD); maturation markers CD83 (HB15e, Biolegend) and CD86 (2331; BD); adhesion marker CD62L (SK11, BD); and migration markers CCR2 (K036C2, Biolegend) and CCR7 (150503: BD); and fixed with 1% paraformaldehyde for flow cytometry. .. Samples were acquired on an LSRFortessa flow cytometer (BD Biosciences) and analysed using FlowJo software v10 (Tree Star).
Migration:Article Title: Human influenza virus infection elicits distinct patterns of monocyte and dendritic cell mobilization in blood and the nasopharynx
Article Snippet: Excess PBMCs were cryopreserved in FBS (Gibco) + 10% DMSO (Sigma) and stored in liquid nitrogen. .. Phenotypic analysis was performed on NPA cells and PBMCs using Live/Dead Blue; lineage markers CD3 (SK7; BD), CD19 (HIB19; Biolegend), CD20 (L27, BD), CD45 (HI30; BD), CD56 (HCD56, BD) and CD66abce (TET2, Miltenyi Biotec); HLA-DR (TU36, Life Technologies), CD14 (M5E2, BD), CD16 (3GE, Biolegend), CD11c (B-Ly6, BD), CD1c (AD5-8E7; Miltenyi), CD141 (AD5-14H12; Miltenyi), CD123 (7G3; BD); maturation markers CD83 (HB15e, Biolegend) and CD86 (2331; BD); adhesion marker CD62L (SK11, BD); and migration markers CCR2 (K036C2, Biolegend) and CCR7 (150503: BD); and fixed with 1% paraformaldehyde for flow cytometry. .. Samples were acquired on an LSRFortessa flow cytometer (BD Biosciences) and analysed using FlowJo software v10 (Tree Star).
Staining:Article Title: Decreased levels and function of dendritic cells in blood and airways predict COVID ‐19 severity
Article Snippet: .. After Live/Dead Blue staining (Invitrogen), cells were incubated with human FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) and then stained with antibodies against surface proteins: CD1c (AD5‐8E7; Miltenyi Biotec), CD3 (SK7; Becton, Dickinson and Company (BD), Franklin Lakes, NJ, USA), CD11c (B‐Ly6; BD), CD14 (M5E2; BD), CD16 (3GE; BioLegend, San Diego, CA, USA), CD19 (HIB19; BioLegend), CD20 (L27; BD), CD45 (HI30; BD), CD56 (HCD56; BD), CD62L (SK11; BD), CD66abce (TET2; Miltenyi Biotec), CD86 (2331; BD), CD123 (7G3; BD), CD141 (AD5‐14H12; Miltenyi Biotec), CCR2 (K036C2; BioLegend), CCR7 (150 503; BD) and HLA‐DR (TU36; Life Technologies, Waltham, MA, USA). .. A subset of respiratory samples were stained with another panel: CD3 (SP34‐2; BBD), CD4 (L200; BD), CD11c (B‐ly6; BD), CD14 (M5E2; BD), CD16 (3G8; BD), CD19 (SJ25‐C1; Thermo Fisher Scientific, Waltham, MA, USA), CD45 (HI30; BD), CD56 (HCD56; BioLegend), CD66abce (TET2; Miltenyi Biotec), CD123 (7G3; BD), LOX‐1 (15C4; BioLegend) and HLA‐DR (L243; BioLegend).
Article Title: Decreased levels and function of dendritic cells in blood and airways predict COVID ‐19 severity
Article Snippet: .. Cells were washed with PBS and stained using Live/Dead Blue (Invitrogen, Thermo Fisher Scientific), incubated with human FcR blocking reagent (Miltenyi Biotec), and stained at 4°C for 20 min with antibodies against the following surface proteins: CD1c (AD5‐8E7; Miltenyi Biotec), CD3 (SK7; BD), CD11c (B‐Ly6; BD), CD14 (M5E2; BD), CD16 (3GE; BioLegend), CD19 (HIB19; BioLegend), CD20 (L27; BD), CD45 (HI30; BD), CD56 (HCD56; BD), CD66abce (TET2; Miltenyi Biotec), CD86 (2331; BD), CD123 (7G3; BD), CD141 (AD5‐14H12; Miltenyi Biotec), and HLA‐DR (TU36; Life Technologies, Thermo Fisher Scientific). .. Cells were fixed and permeabilised with permeabilisation buffer (Thermo Fisher Scientific) and stained with antibodies against IFN‐α (LT27:295, Miltenyi Biotec), IL‐6 (MQ2‐13A5, Thermo Fisher Scientific), and TNF (Mab11, BD) at 4°C for 15 min.
Incubation:Article Title: Decreased levels and function of dendritic cells in blood and airways predict COVID ‐19 severity
Article Snippet: .. After Live/Dead Blue staining (Invitrogen), cells were incubated with human FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) and then stained with antibodies against surface proteins: CD1c (AD5‐8E7; Miltenyi Biotec), CD3 (SK7; Becton, Dickinson and Company (BD), Franklin Lakes, NJ, USA), CD11c (B‐Ly6; BD), CD14 (M5E2; BD), CD16 (3GE; BioLegend, San Diego, CA, USA), CD19 (HIB19; BioLegend), CD20 (L27; BD), CD45 (HI30; BD), CD56 (HCD56; BD), CD62L (SK11; BD), CD66abce (TET2; Miltenyi Biotec), CD86 (2331; BD), CD123 (7G3; BD), CD141 (AD5‐14H12; Miltenyi Biotec), CCR2 (K036C2; BioLegend), CCR7 (150 503; BD) and HLA‐DR (TU36; Life Technologies, Waltham, MA, USA). .. A subset of respiratory samples were stained with another panel: CD3 (SP34‐2; BBD), CD4 (L200; BD), CD11c (B‐ly6; BD), CD14 (M5E2; BD), CD16 (3G8; BD), CD19 (SJ25‐C1; Thermo Fisher Scientific, Waltham, MA, USA), CD45 (HI30; BD), CD56 (HCD56; BioLegend), CD66abce (TET2; Miltenyi Biotec), CD123 (7G3; BD), LOX‐1 (15C4; BioLegend) and HLA‐DR (L243; BioLegend).
Article Title: Decreased levels and function of dendritic cells in blood and airways predict COVID ‐19 severity
Article Snippet: .. Cells were washed with PBS and stained using Live/Dead Blue (Invitrogen, Thermo Fisher Scientific), incubated with human FcR blocking reagent (Miltenyi Biotec), and stained at 4°C for 20 min with antibodies against the following surface proteins: CD1c (AD5‐8E7; Miltenyi Biotec), CD3 (SK7; BD), CD11c (B‐Ly6; BD), CD14 (M5E2; BD), CD16 (3GE; BioLegend), CD19 (HIB19; BioLegend), CD20 (L27; BD), CD45 (HI30; BD), CD56 (HCD56; BD), CD66abce (TET2; Miltenyi Biotec), CD86 (2331; BD), CD123 (7G3; BD), CD141 (AD5‐14H12; Miltenyi Biotec), and HLA‐DR (TU36; Life Technologies, Thermo Fisher Scientific). .. Cells were fixed and permeabilised with permeabilisation buffer (Thermo Fisher Scientific) and stained with antibodies against IFN‐α (LT27:295, Miltenyi Biotec), IL‐6 (MQ2‐13A5, Thermo Fisher Scientific), and TNF (Mab11, BD) at 4°C for 15 min.
Blocking Assay:Article Title: Decreased levels and function of dendritic cells in blood and airways predict COVID ‐19 severity
Article Snippet: .. After Live/Dead Blue staining (Invitrogen), cells were incubated with human FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) and then stained with antibodies against surface proteins: CD1c (AD5‐8E7; Miltenyi Biotec), CD3 (SK7; Becton, Dickinson and Company (BD), Franklin Lakes, NJ, USA), CD11c (B‐Ly6; BD), CD14 (M5E2; BD), CD16 (3GE; BioLegend, San Diego, CA, USA), CD19 (HIB19; BioLegend), CD20 (L27; BD), CD45 (HI30; BD), CD56 (HCD56; BD), CD62L (SK11; BD), CD66abce (TET2; Miltenyi Biotec), CD86 (2331; BD), CD123 (7G3; BD), CD141 (AD5‐14H12; Miltenyi Biotec), CCR2 (K036C2; BioLegend), CCR7 (150 503; BD) and HLA‐DR (TU36; Life Technologies, Waltham, MA, USA). .. A subset of respiratory samples were stained with another panel: CD3 (SP34‐2; BBD), CD4 (L200; BD), CD11c (B‐ly6; BD), CD14 (M5E2; BD), CD16 (3G8; BD), CD19 (SJ25‐C1; Thermo Fisher Scientific, Waltham, MA, USA), CD45 (HI30; BD), CD56 (HCD56; BioLegend), CD66abce (TET2; Miltenyi Biotec), CD123 (7G3; BD), LOX‐1 (15C4; BioLegend) and HLA‐DR (L243; BioLegend).
Article Title: Decreased levels and function of dendritic cells in blood and airways predict COVID ‐19 severity
Article Snippet: .. Cells were washed with PBS and stained using Live/Dead Blue (Invitrogen, Thermo Fisher Scientific), incubated with human FcR blocking reagent (Miltenyi Biotec), and stained at 4°C for 20 min with antibodies against the following surface proteins: CD1c (AD5‐8E7; Miltenyi Biotec), CD3 (SK7; BD), CD11c (B‐Ly6; BD), CD14 (M5E2; BD), CD16 (3GE; BioLegend), CD19 (HIB19; BioLegend), CD20 (L27; BD), CD45 (HI30; BD), CD56 (HCD56; BD), CD66abce (TET2; Miltenyi Biotec), CD86 (2331; BD), CD123 (7G3; BD), CD141 (AD5‐14H12; Miltenyi Biotec), and HLA‐DR (TU36; Life Technologies, Thermo Fisher Scientific). .. Cells were fixed and permeabilised with permeabilisation buffer (Thermo Fisher Scientific) and stained with antibodies against IFN‐α (LT27:295, Miltenyi Biotec), IL‐6 (MQ2‐13A5, Thermo Fisher Scientific), and TNF (Mab11, BD) at 4°C for 15 min.
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