Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: SIRPα and FcγRI are arranged in discrete nanoclusters at macrophage surfaces. (A and B) TIRF and dSTORM images of SIRPα (A) and FcγRI (B) at the surface of human macrophages seeded onto PLL- (nonactivated, top) or hIgG-coated slides (bottom) for 10 min and stained with fluorescently labeled specific antibodies. Bars, 5 µm. Regions delineated by white squares are zoomed-in and shown with corresponding density maps (pseudocolor scale), thresholded binary maps and Ripley’s K analysis of the molecules in the selected regions. Bars, 1 µm. L(r)-r represents the degree of clustering relative to simulated random distributions, indicated by the 99% confidence intervals (CIs); r is the radial scale. (C–E) Nanocluster areas (C), density (D), and percentage of localizations in nanoclusters (E) for SIRPα and FcγRI under nonactivating (black) or hIgG-activating (gray) conditions were calculated by subjecting dSTORM data to spatial point-pattern analysis and thresholding. Each symbol represents the median of several 5 × 5 µm regions from the same cell. Horizontal lines and error bars represent mean ± SD. Data are from a minimum of 30 cells from three independent donors. ns, not significant; ****, P < 0.0001; two-tailed t test assuming unequal variance. (F and G) Label-density variation analysis for SIRPα (F) and FcγRI (G) yields characteristic normalized ρ/η curves for clustered proteins. Cells were stained with anti–SIRPα-AF647 (F) or anti–FcγRI-AF488 (G) at different labeling concentrations and imaged by dSTORM. Each data point represents a single cell, color-coded by antibody concentration used for labeling. Red lines indicate reference curves for a random distribution of molecules.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Staining, Labeling, Two Tailed Test, Concentration Assay

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: SIRPα and FcγRI nanoclusters are constitutively associated in nonactivated human macrophages but segregate upon activation with hIgG. (A) TIRF and dSTORM images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 min on slides coated with PLL (nonactivated, top) or hIgG (middle) and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares (middle column) are shown enlarged (right columns) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. As a positive control, macrophages seeded onto PLL-coated slides were stained with anti–FcγRI-AF488 mAb followed by anti–mouse IgG1-AF647 secondary antibody (bottom). (B) CBC histograms of the single-molecule distributions of the colocalization parameter for SIRPα and FcγRI in cells seeded onto PLL- (gray) or hIgG-coated (red) slides for 10 min or for positive control data (green). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (C) Nearest-neighbor (NN) analysis from data shown in (B). Each symbol represents the median NN of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ****, P < 0.0001; two-tailed t test assuming unequal variance. (D) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥ 20,000 clusters from a minimum of 10 cells per condition) from cells seeded onto PLL- (light gray), hIgG-coated (light red) slides, or positive control data (green). Corresponding simulated data are also shown, in which the centroid positions of SIRPα nanoclusters in both nonactivating (dark gray) and hIgG-activating conditions (dark red) were randomized within the cell area.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Activation Assay, Incubation, Staining, Fluorescence, Positive Control, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: SIRPα and the low-affinity Fc receptor, FcγRII, are segregated on a nanometer scale. (A) TIRF and dSTORM images showing FcγRII (green) and SIRPα (red) at the surface of human macrophages incubated for 10 or 30 min on slides coated with PLL (nonactivated) or hIgG and stained with anti–FcγRII-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (B) CBC histograms of the single-molecule distributions of the colocalization parameter for SIRPα and FcγRII in cells seeded onto PLL- or hIgG-coated slides for 10 (light gray and dark gray, respectively) or 30 min (light red and dark red, respectively) or for positive control data (green). The positive control data in this figure is the same as in . Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (C) NND analysis from data shown in B. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ***, P < 0.001; one-way analysis of variance (ANOVA) with Tukey’s post-hoc test. (D) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). a.u. arbitrary units; NN, nearest neighbor; PC, positive control.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Incubation, Staining, Fluorescence, Positive Control

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: FcγRs reorganize into concentric rings upon activation. (A) TIRF images of FcγRI (top) and FcγRII (bottom) at the surface of human macrophages incubated for 10 or 30 min on slides coated with PLL (nonactivated) or hIgG and stained with fluorescently labeled specific antibodies. Bars, 10 µm. (B) TIRF and dSTORM images of FcγRI (green) and FcγRII (red) at the surface of macrophages incubated for 10 or 30 min on slides coated with PLL or hIgG and stained with anti–FcγRI-AF488 and anti–FcγRII-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and FcγRII in cells seeded onto PLL- or hIgG-coated slides for 10 (light gray and dark gray, respectively) or 30 min (light red and dark red, respectively) or for positive control data (green). The positive control data in this figure are the same as in . Data are from a minimum of 10 cells from three independent donors. Bars represent mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ****, P < 0.0001; one-way ANOVA with Tukey’s post-hoc test. (E) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). a.u., arbitrary units; NN, nearest neighbor; PC, positive control.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Activation Assay, Incubation, Staining, Labeling, Fluorescence, Positive Control

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Specific activation of FcγRI is required for its reorganization into concentric rings and segregation from SIRPα nanoclusters. (A and B) TIRF (bars, 10 µm) and dSTORM (bars, 5 µm) images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 (A) or 30 min (B) on slides coated with hIgG1 or hIgG2 and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα in cells seeded onto hIgG1- or hIgG2-coated slides for 10 (light gray and dark gray, respectively) or 30 min (light red and dark red, respectively). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ****, P < 0.0001; one-way ANOVA with Tukey’s post-hoc test. (E) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). a.u., arbitrary units; NN, nearest neighbor; PC, positive control.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Activation Assay, Incubation, Staining, Fluorescence, Positive Control

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Rearrangement of macrophage surface receptors triggered by mobile hIgG. (A) TIRF images of FcγRI at the surface of human macrophages incubated for 10 min on SLBs loaded with streptavidin (nonactivating) or with streptavidin-hIgG (activating) and stained with a fluorescently labeled specific antibody. Two example images are shown for each condition. Bars, 10 µm. (B) dSTORM images of FcγRI (green) and SIRPα (red) at the surface of macrophages seeded as in A and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares are shown enlarged with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C–E) Nanocluster areas (C), density (D), and percentage of localizations in nanoclusters (E) for SIRPα and FcγRI under nonactivating (black) or hIgG-activating (gray) conditions. Each symbol represents the median of several 5 × 5 µm regions from the same cell. Horizontal lines and error bars represent mean ± SD. Data are from a minimum of 30 cells from two independent experiments. ns, not significant; *, P < 0.05; ****, P < 0.0001; two-tailed t test assuming unequal variance. (F) CBC histograms of the single-molecule distributions of the colocalization parameter for SIRPα and FcγRI in cells seeded as in A. Data are from a minimum of 30 cells from two independent experiments. Bars represent mean ± SD. (G) NND analysis from data shown in F. Each symbol represents median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ****, P < 0.0001; two-tailed t test assuming unequal variance. (H) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥10,000 clusters from a minimum of 10 cells per condition) from cells seeded onto control nonactivating (light gray) or hIgG-loaded activating (light red) SLBs.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Incubation, Staining, Labeling, Fluorescence, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Ligation of SIRPα impairs the reorganization of surface FcγRI. (A) Human macrophages were incubated for 24 h in wells coated with PLL, 20 µg/ml of hCD47, or with increasing concentrations of hCD47 in the presence of 10 µg/ml of hIgG, as indicated. M-CSF release was assessed by ELISA. Bars represent mean ± SD from three donors. Each color represents one individual donor. (B) TIRF images of FcγRI at the surface of human macrophages incubated for 10 min on slides coated with hCD47 or hCD47 plus hIgG and stained with fluorescently labeled specific antibody. Bars, 10 µm. (C) TIRF and dSTORM images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 min on slides coated with hCD47 (top) or hCD47 plus hIgG (bottom) and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (D and G) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα (D) and for FcγRI and pSHP-1 Y536 (G) in cells seeded onto slides coated with PLL (light gray), hCD47 (light red), hCD47 plus hIgG (dark red), or hIgG (dark gray) for 10 (D) or 5 min (G). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (E and H) NND analysis from data shown in D and G, respectively. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA with Tukey’s post-hoc test. (F and I) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). Graphs compare colocalization between FcγRI and SIRPα (F) and FCγRI and pSHP-1 Y536 (I). a.u., arbitrary units; NN, nearest neighbor; PC, positive control.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Ligation, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Labeling, Fluorescence, Positive Control

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Segregation and reorganization of FcγRI is dependent on the actin cytoskeleton and formins, but not myosin II. (A) TIRF image of FcγRI (white; bars, 20 µm) and dSTORM images (bars, 5 µm) of FcγRI (green) and SIRPα (red) at the surface of human macrophages pretreated with 1 µM latrunculin A, 0.5 µM jasplakinolide, 10 µM blebbistatin or 10 µM SMIFH2. Cells were then seeded onto slides coated with PLL (nonactivated) or hIgG for 10 min, and stained with anti-FcγRI-AF488 and anti-SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column). Bars, 1 µm. (B) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα in cells pretreated with drugs as indicated and seeded onto slides coated with PLL (gray) or hIgG (latrunculin A [Lat A], dark gray; jasplakinolide [Jasp], red; SMIFH2, green; or blebbistatin [Bleb], blue) for 10 min. Data are from a minimum of 30 cells per condition from three independent donors. Bars represent mean ± SD. (C) NND analysis from data shown in B. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ****, P < 0.0001; two-tailed t test assuming unequal variance. (D) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). (E–G) Nanocluster areas (E), density (F), and percentage of localizations in nanoclusters (G) for SIRPα and FcγRI under nonactivating (black) or hIgG-activating (gray) conditions after pretreatment of cells with blebbistatin or DMSO control. Each symbol represents the median of several 5 × 5 µm regions from the same cell. Horizontal lines and error bars represent mean ± SD. Data are from a minimum of 30 cells from three independent donors. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; two-tailed t test assuming unequal variance. NN, nearest neighbor.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Staining, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Src-family kinase signaling, but not Syk or PI3K signaling, is indispensable for reorganization of macrophage surfaces. (A) Immunoblots of phosphorylated AKT in nonactivated (PLL) or hIgG-activated human macrophages pretreated with vehicle (DMSO), as a control, 10 µM PP2 (left), 100 µM piceatannol (PCT; middle), or 1 µM wortmannin (Wort; right). Blots represent two independent experiments. (B) TIRF image of FcγRI (white; bars, 20 µm) and dSTORM images (bars, 5 µm) of FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated with vehicle (DMSO), PP2, PCT, or Wort, pretreated as in A. Cells were then seeded onto slides coated with PLL (nonactivated) or hIgG for 10 min and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column). Bars, 1 µm. (C) CBC histograms for FcγRI and SIRPα in cells pretreated as in A and seeded onto slides coated with PLL (gray) or hIgG (DMSO, dark gray; PP2, red; PCT, green; and Wort, blue) for 10 min, as indicated. Data are from a minimum of 30 cells from three independent donors. Bars show mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; ****, P < 0.0001; one-way ANOVA with Tukey’s post-hoc test. (E) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition).

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Western Blot, Incubation, Staining

Journal: bioRxiv

Article Title: c-MAF dependent perivascular macrophages regulate diet induced metabolic syndrome

doi: 10.1101/2021.02.07.430147

Figure Lengend Snippet: (A) Flow cytometry analysis of cell surface markers expressed by VAMs in adult WT C57BL/6 mice. eWAT VAMs were gated as CD45 + Lin - CD11b + CD64 + CD206 HIGH . The magenta and blue gates correspond to VAM2 and VAM1, respectively. (B) Flow cytometry analysis of human visceral fat macrophages. Percentage of cells is shown. Representative dot plot. n=3. (C) VAMs are highly endocytic in vivo . Ovalbumin-A647 (Ova-A647) was injected i.v. and after the depicted time the uptake of Ovalbumin was measured by flow cytometry. VAMs gated as mentioned in (A). Representative histograms; n = 3. Dashed lines represent the median fluorescence intensity of the VAM2. Bar graphs show the fold increase of the median ovalbumin-A647 fluorescence intensity after injection normalized to the individual autofluorescence of each subpopulation, n = 3. CD11b + DCs gated as CD45 + Lin - CD11b + CD64 - CD11c + MHCII + . Bar graphs display mean±SEM. (D) Lyve1 Cre Maf F/F mice do not show abnormality in liquid drainage from extremities. Foot pad were not swollen in these animals. Representative figure n=5. (E) Lyve1 Cre Maf F/F mice display functional lymphatic vessels. Evans blue dye (40mg/ml) was injected in one hind foot pad of Lyve1 Cre Maf F/F or littermate controls and the dye drainage through lymphatic vessels around the small saphenous vein was visualized 30 min post injection. Blue arrowheads are indicating evans blue loaded lymphatic vessels. Representative figure. n=3 for each group. (F) Evans blue dye is efficiently drained into the Popliteal lymph node of Lyve1 Cre Maf F/F . Lymph nodes of the experiment depicted in (E) are shown. pLN = Popliteal lymph node. Scale Bars, 500 μm. Representative figure. n=3 for each group. (G) Lyve1 Cre Maf F/F mice display a preserved capacity to form a lymphatic vessel tree within the eWAT. Whole eWAT fat pad were clarified, stained with LYVE1 (Orange) and imaged using a light sheet microscope. Scale Bars, 300 μm. 4x magnification. (H) Lymphatic vessels of Lyve1 Cre Maf F/F display a similar structure to those observed in littermate controls. Clarified eWAT were stained with LYVE1 (Orange) and imaged using a confocal microscope. Scale Bars, 20 μm. 20x magnification. Representative images. (I) Distribution of myeloid cells per gram of eWAT in the different conditional knockout models used in main . Graph bar colors correspond to the models listed in main . For each model, littermate controls were used as Maf F/F WT controls. Ly6C LOW Mon: Ly6C LOW Monocytes were gated as CD45 + Lin - CD11b + CX3CR1 + MHCII - Ly6C LOW . Neutrophils were gated as CD45 + CD11b + MHCII - Ly6G + . Eosinophils were gated as CD45 + CD11b + MHCII - SSC HIGH SiglecF + . PreVAMs were gated as CD45 + Lin - CD11b + CD206 LOW/INT MHCII + CD64 + CD11c - . DPs: Double positive macrophages were gated as CD45 + Lin - CD11b + CD206 LOW/INT MHCII + CD64 + CD11c + . CD11b - DCs were gated as CD45 + Lin - CD11b - CD64 - CD11c + MHCII + . Ptn: Protein. Lin: CD90 + CD19 + Ly6G + SiglecF + .

Article Snippet: Jackson Immunoresearch : Donkey Anti-Rat A647 (712-605-153); Donkey Anti-Rat BV421 (712-675-153), Donkey Anti-Goat A488 (705-545-147); Donkey Anti-Goat A647 (705-605-147); Normal Donkey Serum (017-000-121).

Techniques: Flow Cytometry, In Vivo, Injection, Fluorescence, Functional Assay, Staining, Microscopy, Knock-Out

Journal: bioRxiv

Article Title: c-MAF dependent perivascular macrophages regulate diet induced metabolic syndrome

doi: 10.1101/2021.02.07.430147

Figure Lengend Snippet: (A) Flow cytometry gate strategy used to analyze brain perivascular macrophages. Percentage of cells is shown. (B) Flow cytometry analysis of cell surface markers expressed by BAMs. Histograms represent the pink and blue gates depicted in the BAMs dot plot in (A). Representative histograms of at least n=5. Dashed lines represent the median fluorescence intensity of microglia as a comparative measure in the organ. (C) BAMs CD206 HIGH express lower levels of CD45 in relation to bone marrow derived monocytes. n≥5. Monocytes were gated as CD45 + Lin - CD11b + CX3CR1 + MHCII - Ly6C HIGH . (D) BAMs CD206 HIGH are highly endocytic in vivo . Ovalbumin-A647 (Ova-A647) was injected i.v. and after the depicted time the uptake of Ovalbumin was measured by flow cytometry. BAMs gated as mentioned in (A). Representative histograms; n = 3. Dashed lines represent the median fluorescence intensity of the BAMs CD206 HIGH . Bar graphs show the fold increase of the median ovalbumin-A647 fluorescence intensity after injection normalized to the individual autofluorescence of each subpopulation, n = 3. Bar graphs display mean±SEM. (E) Neonate Brains (P5) show early ablation of BAMs CD206 HIGH . Total Brain cell numbers in Csf1r Cre Maf F/F . Littermate controls were used as Maf F/F WT controls. n=3. Representative of 2 independent experiments. (F) Distribution of myeloid cells per brain in the different conditional knockout models used in main . For each model, littermate controls were used as Maf F/F WT controls. (G) Confocal image of clarified brains of Lyve1 Cre Maf F/F and littermate control mice demonstrating the ablation of BAMs CD206 HIGH . Brains were stained with anti-CD206 (Green), anti-LYVE1 (Cyan) and the background (White). Scale Bars, 20 μm. 20x magnification. Representative images. n=3. Ly6C LOW Mon: Ly6C LOW Monocytes were gated as CD45 + Lin - CD11b + CX3CR1 + MHCII - Ly6C LOW . Neutrophils were gated as CD45 + CD11b + MHCII - Ly6G + . CD11b - DCs were gated as CD45 + Lin - CD11b - CD64 - CD11c + MHCII + . CD11b + DCs were gated as CD45 + Lin - CD11b + CD64 - CD11c + MHCII + . Ptn: Protein. Lin: CD90 + CD19 + Ly6G + SiglecF + .

Article Snippet: Jackson Immunoresearch : Donkey Anti-Rat A647 (712-605-153); Donkey Anti-Rat BV421 (712-675-153), Donkey Anti-Goat A488 (705-545-147); Donkey Anti-Goat A647 (705-605-147); Normal Donkey Serum (017-000-121).

Techniques: Flow Cytometry, Fluorescence, Derivative Assay, In Vivo, Injection, Knock-Out, Control, Staining

Journal: bioRxiv

Article Title: c-MAF dependent perivascular macrophages regulate diet induced metabolic syndrome

doi: 10.1101/2021.02.07.430147

Figure Lengend Snippet: (A) Flow cytometry gate strategy used to analyze large intestine (Li) macrophages of mice used in this study. Percentage of cells is shown. (B) Flow cytometry analysis of cell surface markers expressed by VAMs from Li. Histograms represent the magenta, blue and green gates depicted in the Li macrophages in (A). CD38 - Mac: CD38 - macrophages were gate as CD45 + CD11b + CD64 + CD38 - MHCII + . Mon: Monocytes were gated as CD45 + Lin - CD11b + CX3CR1 + MHCII - Ly6C HIGH . Representative histograms of at least n=5. Dashed lines represent the median fluorescence intensity of monocytes as a comparative measure in the organ. (C) VAMs Li are also localized in close proximity to enteric-associated neurons in the large intestine myenteric plexus of adult WT mice. Colon stained with anti-CD206 (Green), anti-ANNA-1 (Magenta – neuron body) and anti-MHClI (White). Scale Bars, 20 μm. 20x magnification. n = 3. (D) Same as in c in higher details. Figure depicts the white box shown in (C) showing in details the close proximity of perivascular macrophages to enteric-associated neurons. Scale Bars, 10 μm. 20x magnification. (E) The small intestine muscularis layer also display VAM related macrophages (pink) and non-related (green) perivascular macrophages. Cells were gated as depicted in (A). Percentage of cells is shown. n=3. (F) VAMs Li are highly endocytic in vivo . Ovalbumin-A647 (Ova-A647) was injected i.v. and after the depicted time the uptake of Ovalbumin was measured by flow cytometry. VAMs Li gated as mentioned in (A). Representative histograms; n = 3. Dashed lines represent the median fluorescence intensity of the VAM2 Li. Bar graphs show the fold increase of the median ovalbumin-A647 fluorescence intensity after injection normalized to the individual autofluorescence of each subpopulation, n = 3. CD11b + DCs gated as CD45 + Lin - CD11b + CD64 - CD11c + MHCII + . Bar graphs display mean±SEM. (G) Distribution of myeloid cells per colon in the different conditional knockout models used in main and . For each model, littermate controls were used as Maf F/F WT controls. (H) Representative dot plot demonstrating the impact of Maf deletion in the small intestine muscularis layer of animals Csf1r Cre Maf F/F . Each dot in the bar graphs represents one animal. Bar graphs display mean values. Ly6C LOW Mon: Ly6C LOW Monocytes were gated as CD45 + Lin - CD11b + CX3CR1 + MHCII - Ly6C LOW . Neutrophils were gated as CD45 + CD11b + MHCII - Ly6G + . Eosinophils were gated as CD45 + CD11b + MHCII - SSC HIGH SiglecF + . CD11b - DCs were gated as CD45 + Lin - CD11b - CD64 - CD11c + MHCII + . CD11b + DCs were gated as CD45 + Lin - CD11b + CD64 - CD11c + MHCII + . Mac: Macrophage. Ptn: Protein. Lin: CD90 + CD19 + Ly6G + SiglecF + .

Article Snippet: Jackson Immunoresearch : Donkey Anti-Rat A647 (712-605-153); Donkey Anti-Rat BV421 (712-675-153), Donkey Anti-Goat A488 (705-545-147); Donkey Anti-Goat A647 (705-605-147); Normal Donkey Serum (017-000-121).

Techniques: Flow Cytometry, Fluorescence, Staining, In Vivo, Injection, Knock-Out

Journal: bioRxiv

Article Title: c-MAF dependent perivascular macrophages regulate diet induced metabolic syndrome

doi: 10.1101/2021.02.07.430147

Figure Lengend Snippet: (A) Confocal images of clarified eWAT from animals Lyve1 Cre Maf F/F , LySM Cre Maf F/F , Csf1r Cre Maf F/F and littermate control stained with anti-CD31 (Magenta). Scale Bars, 20 μm. 20x magnification. Representative images. n≥3. (B) Quantification of the vascular surface area assessed by the anti-CD31 staining observed in (A) Three to four 50μm deep images were taken per animal and the vascular surface area reconstructed using the IMARIS software. Each dot represents the quantification for each picture. n≥3. Representative of 3 independent experiments. (C) Quantification of the vascular surface area assessed by the anti-CD31 staining of young 5 weeks old Csf1r Cre Maf F/F mice. Three to four 50μm deep images were taken per animal and the vascular surface area reconstructed using the IMARIS software. Each dot represents the quantification for each picture. n=3. (D) Evaluation of the endocytic capacity of eWAT VAMs in the different animal models used in (A) Ovalbumin-A647 (Ova-A647) was injected i.v. and after the depicted time the uptake of ovalbumin was measured by flow cytometry in the eWAT. Representative histograms; n = 3. The dashed line represents the median fluorescence intensity of the WT VAMs. (E) Distribution of eWAT macrophages per gram of adipose tissue in Lyve1 Cre Csf1r F/F mice. Littermate controls were used as Csf1r F/F WT controls. Each dot in the bar graphs represents one animal. Representative of 3 independent experiments. (F) Lyve1 Cre Csf1r F/F mice display increased vascular density in the eWAT. Confocal images of clarified eWAT stained with anti-CD31 (Magenta). Scale Bars, 20 μm. 20x magnification. Representative images. n≥3. (G) Quantification of the vascular surface area assessed by the anti-CD31 staining observed in (F). Three to four 50μm deep images were taken per animal and the vascular surface area reconstructed using the IMARIS software. Each dot represents the quantification for each picture. n≥3. Representative of 2 independent experiments. Bar graphs display mean values. See also .

Article Snippet: Jackson Immunoresearch : Donkey Anti-Rat A647 (712-605-153); Donkey Anti-Rat BV421 (712-675-153), Donkey Anti-Goat A488 (705-545-147); Donkey Anti-Goat A647 (705-605-147); Normal Donkey Serum (017-000-121).

Techniques: Control, Staining, Software, Injection, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: c-MAF dependent perivascular macrophages regulate diet induced metabolic syndrome

doi: 10.1101/2021.02.07.430147

Figure Lengend Snippet: (A) Experimental design for (B-H). (B) Glucose tolerance test (GTT) of Csf1r Cre Maf F/F mice and littermate controls under normal or high fat diet (ND or HFD). n>3. Representative of 3 independent experiments. (C) Area under the curve of the GTT displayed in (B) (Arbitrary units). Bar graphs display mean±SEM. (D, E) Animal weight and eWAT fat pads weight of animals depicted in (B). (F) Evaluation of eWAT VAMs endocytic capacity in animals submitted to HFD. Ovalbumin-A647 (Ova-A647) was injected i.v. and after the depicted time the uptake of ovalbumin was measured by flow cytometry in the eWAT. Representative histograms; n ≥4. The dashed line represents the median fluorescence intensity of macrophages from animals under ND regimen. (G) Fold increase of the median ovalbumin-A647 fluorescence intensity after injection as depicted in (F) normalized to the individual autofluorescence of each subpopulation, n≥4. Each dot in the bar graphs represents one animal. Bar graphs display mean values. (H) Distribution of VAMs and DPs (CD11c + CD64 + ) macrophages per gram of eWAT in the animals depicted in (B). Each dot in the bar graphs represents one animal. Bar graphs display mean values. DPs: Double positive macrophages were gated as CD45 + Lin - CD11b + CD206 LOW/INT MHCII + CD64 + CD11c + .See also .

Article Snippet: Jackson Immunoresearch : Donkey Anti-Rat A647 (712-605-153); Donkey Anti-Rat BV421 (712-675-153), Donkey Anti-Goat A488 (705-545-147); Donkey Anti-Goat A647 (705-605-147); Normal Donkey Serum (017-000-121).

Techniques: Injection, Flow Cytometry, Fluorescence

Journal: Nature Communications

Article Title: Encoding extracellular modification of artificial cell membranes using engineered self-translocating proteins

doi: 10.1038/s41467-024-53783-4

Figure Lengend Snippet: a General scheme for testing translocation of peptides by GUV antibody binding assay. A Cy5-conjugated monoclonal antibody specific for a given peptide insert is encapsulated into GUVs. αHL monomers with the respective insert are added to the outside of the GUVs. Upon external treatment of GUVs with αHL-GFP fusion proteins containing the peptide insert, the antibody will localize to the membrane, indicating that the peptide insert has fully translocated across the membrane and can be bound by the internal antibody. b GUVs encapsulated with Cy5 modified anti-6XHis-tag antibodies before and after treatment with αHL-GFP containing the L-6XHis-L loop insert. The GUV membrane shows increased Cy5 (antibody) signal after treatment. Scale bar: 10 µm. c A larger population of GUVs encapsulating the Cy5 anti-6XHis-tag antibody, all showing increased membrane fluorescence after treatment with αHL-GFP containing the L-6XHis-L loop insert. Scale bar: 10 µm. d Cy5 antibody assays with all the peptide inserts that were shown to form functional pores. Scale bar: 10 µm. e Quantitative analysis of the antibody binding assay. The ratio of membrane fluorescence to lumen fluorescence after translocation of the peptides shown in d is plotted (further information in Supplementary Fig. ). Untreated GUVs show a mean fluorescence ratio of 82%, whereas treatment with hemolysin constructs leads to a fluorescence ratio increase of at least 50%. Data ( n = 8 GUVs per treatment group) was analyzed using an independent t -test (two-tailed). Statistical significance was tested for each treatment group against the untreated group. p < 0.0001 for L-6XHis-L; p < 0.0001 for L 2 -6XHis-L 2 ; p = 0.0135 for L 3 -6XHis-L 3 ; p = 0.0006 for L-Somatostatin-L; p = 0.0005 for L 2 -GLP1-L 2 . Source data are provided as a Source Data file.

Article Snippet: AF® 647-Anti-GLP1 antibody was purchased from Bioss (Cat# bsm-0933M-A647).

Techniques: Translocation Assay, Binding Assay, Membrane, Modification, Fluorescence, Functional Assay, Construct, Two Tailed Test

Journal: Nature Communications

Article Title: Encoding extracellular modification of artificial cell membranes using engineered self-translocating proteins

doi: 10.1038/s41467-024-53783-4

Figure Lengend Snippet: a Cryo-EM image of a representative lipid vesicle after external treatment with αHL containing the L 2 -GLP1-L 2 loop insert. Red arrow: Side-view of αHL cap. Blue arrow: Top view of αHL cap. Scale bar: 10 nm. b 2D class averages of cryo-EM images of αHL containing the L 2 -GLP1-L 2 loop insert in a lipid bilayer. Top view shows the heptameric pore structure. c Lipid bilayer channel recordings of αHL containing the L-6XHis-L loop insert showing pore blockage events. d Lipid bilayer channel recording of αHL containing the L 2 -GLP1-L 2 loop insert. Increasing the length of the flexible linker increases the frequency of pore blockage events. Mean conductance values for αHL with the L-6XHis-L and αHL with the L 2 -GLP1-L 2 are 3.25 nS ± 0.125 nS (50 mV; n = 3) and 1.67 nS ± 0.109 nS (50 mV; n = 3) respectively. Scale bar: 1 s.

Article Snippet: AF® 647-Anti-GLP1 antibody was purchased from Bioss (Cat# bsm-0933M-A647).

Techniques: Cryo-EM Sample Prep

Journal: Stem Cell Research & Therapy

Article Title: AQP0 is a novel surface marker for deciphering abnormal erythropoiesis

doi: 10.1186/s13287-021-02343-4

Figure Lengend Snippet: Characterization of mouse erythropoiesis stages using three surface markers (CD71, TER119, and Aqp0). Flow cytometry analysis of mouse erythropoiesis ( a ). Mouse bone marrow cells were isolated and stained with anti-CD71 antibodies conjugated with FITC, anti-TER119 antibodies conjugated with PE/Cy7, and anti-Aqp0 antibodies conjugated with Alexa Fluor 647. The percentages of positive cells of each antibody in unstained control were showed on the top. CD71 −/dim /TER119 −/dim cells were gated as nonerythroid cells. CD71 high /TER119 low , CD71 high /TER119 high , CD71 dim /TER119 high , and CD71 − /TER119 high were defined as region 1 (R1), region 2 (R2), region 3 (R3), and region 4 (R4), respectively. R3 was gated, and the relationship between FSC (cell size) and Aqp0 expression was analyzed. The percentages of Aqp0 + cells in each population were analyzed and quantified ( b ). Data are representative of four independent experiments and reported as the mean ± standard deviation

Article Snippet: Cells were then stained with anti-CD71 antibodies conjugated with FITC, anti-TER119 antibodies conjugated with PE/Cy7 (BioLegend), and anti-Aqp0 antibodies conjugated with AF647 (Bioss) for 1 h at room temperature.

Techniques: Flow Cytometry, Isolation, Staining, Expressing, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: AQP0 is a novel surface marker for deciphering abnormal erythropoiesis

doi: 10.1186/s13287-021-02343-4

Figure Lengend Snippet: Relative mRNA expressions of erythroid specific transcription factors in each erythroid differentiation stage. Mouse bone marrow cells were isolated and categorized as R1, R2, R3/Aqp0 − , R3/Aqp0 + , R4, and nonerythroid populations on the basis of the fluorescence intensity of three erythroid specific cell surface markers (CD71, TER119, and Aqp0). The mRNA expression level of each gene in the R1 region was normalized to one-fold. Relative folds of mRNA expression of Klf1 ( a ), Nfe2 ( b ), and Gfi1b ( c ) in each cell population relative to R1 were analyzed using a quantitative reverse polymerase chain reaction assay. Data are representative of three independent experiments and reported as the mean ± standard deviation. * P < 0.05 and ** P < 0.01 relative to R1 groups

Article Snippet: Cells were then stained with anti-CD71 antibodies conjugated with FITC, anti-TER119 antibodies conjugated with PE/Cy7 (BioLegend), and anti-Aqp0 antibodies conjugated with AF647 (Bioss) for 1 h at room temperature.

Techniques: Isolation, Fluorescence, Expressing, Polymerase Chain Reaction, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: AQP0 is a novel surface marker for deciphering abnormal erythropoiesis

doi: 10.1186/s13287-021-02343-4

Figure Lengend Snippet: Characterization of human erythropoiesis stages using three surface markers (CD71, CD235a, and AQP0). Flow cytometry analysis of human erythropoiesis ( a ). Human bone marrow cells were isolated and stained with anti-CD71 antibodies conjugated with PE/Cy7, anti-CD235a antibodies conjugated with FITC, and anti-AQP0 antibodies conjugated with Alexa Fluor 647. The percentages of positive cells of each antibody in unstained control were showed on the top. CD71 −/dim /CD235a −/dim cells were gated as nonerythroid cells. CD71 high /CD235a low , CD71 high /CD235a high , CD71 dim /CD235a high , and CD71 − /CD235a high were defined as region 1 (R1), region 2 (R2), region 3 (R3), and region 4 (R4), respectively. R3 was gated, and the relationship between FSC (cell size) and AQP0 expression was analyzed. The percentages of AQP0 high cells in each population were analyzed and quantified ( b ). The percentages of AQP0 low and AQP0 high cells in R3 were shown ( c ). The data are representative of three independent experiments and reported as the mean ± standard deviation

Article Snippet: Cells were then stained with anti-CD71 antibodies conjugated with FITC, anti-TER119 antibodies conjugated with PE/Cy7 (BioLegend), and anti-Aqp0 antibodies conjugated with AF647 (Bioss) for 1 h at room temperature.

Techniques: Flow Cytometry, Isolation, Staining, Expressing, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: AQP0 is a novel surface marker for deciphering abnormal erythropoiesis

doi: 10.1186/s13287-021-02343-4

Figure Lengend Snippet: Characterization of erythropoiesis of patients using three surface markers (CD71, CD235a, and AQP0). Clinical complete blood count values of nine patients ( a ). The bone marrow cells from nine patients were isolated and stained with anti-CD71 antibodies conjugated with PE/Cy7, anti-CD235a antibodies conjugated with FITC, and anti-AQP0 antibodies conjugated with Alexa Fluor 647. CD71 −/dim /CD235a −/dim cells were gated as nonerythroid cells. CD71 high /CD235a low , CD71 high /CD235a high , CD71 dim /CD235a high , and CD71 − /CD235a high were defined as region 1 (R1), region 2 (R2), region 3 (R3), and region 4 (R4), respectively. R3 was gated, and the relationship between FSC (cell size) and AQP0 expression was analyzed. The percentages of AQP0 high cells in each population were analyzed and quantified in patient 1 ( b ), patient 2 ( c ), patient 3 ( d ), patient 4 ( e ), patient 5 ( f ), patient 6 ( g ), patient 7 ( h ), patient 8 ( i ), and patient 9 ( j ). The data are representative of one independent experiment

Article Snippet: Cells were then stained with anti-CD71 antibodies conjugated with FITC, anti-TER119 antibodies conjugated with PE/Cy7 (BioLegend), and anti-Aqp0 antibodies conjugated with AF647 (Bioss) for 1 h at room temperature.

Techniques: Isolation, Staining, Expressing

Journal: Stem Cells International

Article Title: Effects of Delta-Like Noncanonical Notch Ligand 1 Expression of Human Fetal Liver Hepatoblasts on Hematopoietic Progenitors

doi: 10.1155/2019/7916275

Figure Lengend Snippet: Expression of Dlk1 in the human fetal liver. Hepatoblasts of human fetal liver sections were stained for Dlk1 (green) and alpha-fetoprotein (red); cell nuclei were stained with DAPI (blue). Confocal fluorescence microscopy, scale bar: 50 μ m.

Article Snippet: Antibodies used were mouse IgG1 and 2b lineage cocktail (Lin)-FITC, mouse IgG1 CD34-PE, mouse IgG1 CD38-AF700, mouse IgG1 CD31-BV421, mouse IgG1 CD45-BUV395 (all Becton Dickinson), mouse IgG1 CD326-PE (Miltenyi Biotec), and rabbit IgG Dlk1-AF647 (Bioss Antibodies, Woburn, MA); isotypes were mouse IgG1&2b-FITC, IgG1-PE, IgG1-AF700, IgG1-BV421, IgG1-BUV395 (all Becton Dickinson), and rabbit IgG-AF647 (Bioss Antibodies).

Techniques: Expressing, Staining, Fluorescence, Microscopy

Journal: Stem Cells International

Article Title: Effects of Delta-Like Noncanonical Notch Ligand 1 Expression of Human Fetal Liver Hepatoblasts on Hematopoietic Progenitors

doi: 10.1155/2019/7916275

Figure Lengend Snippet: Gene expression of human fetal liver cells after DLK1 knockdown. Total human fetal liver cells were cultured for three (dark grey bars) or five (light grey bars) days with DLK1-targeting siRNA and analyzed for their expression of the hepatic, hematopoietic, endothelial, and cell cycle genes. Gene expression is given as percent of the respective control (100%) that received nontargeting control siRNA. Data are given as means from n = 3 different repeats ± standard deviation. ∗ , ∗∗ , and ∗∗∗ indicate statistically significant differences ( p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively). Abbreviations: AFP: alpha-fetoprotein; CCNE1: cyclin E1; CD34: cluster of differentiation 34; DLK1: delta-like noncanonical Notch ligand 1; EPCAM: epithelial cell adhesion molecule, CD326; GYPA: glycophorin A, CD235a; KRT19: keratin 19, type 1, cytokeratin 19; MKI67: marker of proliferation Ki-67; PECAM1: platelet and endothelial cell adhesion molecule 1, CD31; PTPRC: protein tyrosine phosphatase, receptor type C, CD45; VWF: von Willebrand factor.

Article Snippet: Antibodies used were mouse IgG1 and 2b lineage cocktail (Lin)-FITC, mouse IgG1 CD34-PE, mouse IgG1 CD38-AF700, mouse IgG1 CD31-BV421, mouse IgG1 CD45-BUV395 (all Becton Dickinson), mouse IgG1 CD326-PE (Miltenyi Biotec), and rabbit IgG Dlk1-AF647 (Bioss Antibodies, Woburn, MA); isotypes were mouse IgG1&2b-FITC, IgG1-PE, IgG1-AF700, IgG1-BV421, IgG1-BUV395 (all Becton Dickinson), and rabbit IgG-AF647 (Bioss Antibodies).

Techniques: Expressing, Cell Culture, Standard Deviation, Marker

Journal: Stem Cells International

Article Title: Effects of Delta-Like Noncanonical Notch Ligand 1 Expression of Human Fetal Liver Hepatoblasts on Hematopoietic Progenitors

doi: 10.1155/2019/7916275

Figure Lengend Snippet: Total cell numbers of human fetal liver cells after DLK1 knockdown. Total human fetal liver cells were cultured for three and five days with DLK1-targeting siRNA (light grey bars) or nontargeting control siRNA (black bars), and total cell numbers were determined. Data are given as means from n = 3 biological repeats ± standard deviation. ∗ indicates a statistically significant difference ( p ≤ 0.05).

Article Snippet: Antibodies used were mouse IgG1 and 2b lineage cocktail (Lin)-FITC, mouse IgG1 CD34-PE, mouse IgG1 CD38-AF700, mouse IgG1 CD31-BV421, mouse IgG1 CD45-BUV395 (all Becton Dickinson), mouse IgG1 CD326-PE (Miltenyi Biotec), and rabbit IgG Dlk1-AF647 (Bioss Antibodies, Woburn, MA); isotypes were mouse IgG1&2b-FITC, IgG1-PE, IgG1-AF700, IgG1-BV421, IgG1-BUV395 (all Becton Dickinson), and rabbit IgG-AF647 (Bioss Antibodies).

Techniques: Cell Culture, Standard Deviation

Journal: Stem Cells International

Article Title: Effects of Delta-Like Noncanonical Notch Ligand 1 Expression of Human Fetal Liver Hepatoblasts on Hematopoietic Progenitors

doi: 10.1155/2019/7916275

Figure Lengend Snippet: Flow cytometry analysis of human fetal liver cell cultures after DLK1 knockdown. Total human fetal liver cells cultured with DLK1-targeting siRNA (grey bars) or nontargeting control siRNA (black bars). Cells were analyzed for expression of hematopoietic CD45, lineage (Lin) surface antigens, CD34, CD31, and Lin − CD34 + CD38 − (hematopoietic stem cells). Data are given as means ± standard deviation from n = 3 biological repeats. Abbreviations: fsc: forward scatter; ssc: side scatter; CD: cluster of differentiation.

Article Snippet: Antibodies used were mouse IgG1 and 2b lineage cocktail (Lin)-FITC, mouse IgG1 CD34-PE, mouse IgG1 CD38-AF700, mouse IgG1 CD31-BV421, mouse IgG1 CD45-BUV395 (all Becton Dickinson), mouse IgG1 CD326-PE (Miltenyi Biotec), and rabbit IgG Dlk1-AF647 (Bioss Antibodies, Woburn, MA); isotypes were mouse IgG1&2b-FITC, IgG1-PE, IgG1-AF700, IgG1-BV421, IgG1-BUV395 (all Becton Dickinson), and rabbit IgG-AF647 (Bioss Antibodies).

Techniques: Flow Cytometry, Cell Culture, Expressing, Standard Deviation

Journal: Stem Cells International

Article Title: Effects of Delta-Like Noncanonical Notch Ligand 1 Expression of Human Fetal Liver Hepatoblasts on Hematopoietic Progenitors

doi: 10.1155/2019/7916275

Figure Lengend Snippet: Colony forming unit assays after DLK1 knockdown. Total human fetal liver cells were cultured for five days with DLK1-targeting siRNA (grey bars) or nontargeting control siRNA (black bars). CFU assays were carried out, and hematopoietic colonies were identified in light microscopy. Data are given as means ± standard deviation from n = 3 biological repeats. ∗ indicates a statistically significant difference ( p ≤ 0.05).

Article Snippet: Antibodies used were mouse IgG1 and 2b lineage cocktail (Lin)-FITC, mouse IgG1 CD34-PE, mouse IgG1 CD38-AF700, mouse IgG1 CD31-BV421, mouse IgG1 CD45-BUV395 (all Becton Dickinson), mouse IgG1 CD326-PE (Miltenyi Biotec), and rabbit IgG Dlk1-AF647 (Bioss Antibodies, Woburn, MA); isotypes were mouse IgG1&2b-FITC, IgG1-PE, IgG1-AF700, IgG1-BV421, IgG1-BUV395 (all Becton Dickinson), and rabbit IgG-AF647 (Bioss Antibodies).

Techniques: Cell Culture, Light Microscopy, Standard Deviation

Journal: Stem Cells International

Article Title: Effects of Delta-Like Noncanonical Notch Ligand 1 Expression of Human Fetal Liver Hepatoblasts on Hematopoietic Progenitors

doi: 10.1155/2019/7916275

Figure Lengend Snippet: Cell numbers after Dlk1 + cell depletion. Numbers of Dlk1-negative human fetal liver-derived cells after five days in culture with Dlk1 + cells in direct contact (black bars), without Dlk1 + cells (white bars), or with Dlk1 + cells in inserts (grey bars). Data are given as means from n = 4 biological repeats ± standard deviation. ∗ indicates a statistically significant difference ( p ≤ 0.05).

Article Snippet: Antibodies used were mouse IgG1 and 2b lineage cocktail (Lin)-FITC, mouse IgG1 CD34-PE, mouse IgG1 CD38-AF700, mouse IgG1 CD31-BV421, mouse IgG1 CD45-BUV395 (all Becton Dickinson), mouse IgG1 CD326-PE (Miltenyi Biotec), and rabbit IgG Dlk1-AF647 (Bioss Antibodies, Woburn, MA); isotypes were mouse IgG1&2b-FITC, IgG1-PE, IgG1-AF700, IgG1-BV421, IgG1-BUV395 (all Becton Dickinson), and rabbit IgG-AF647 (Bioss Antibodies).

Techniques: Derivative Assay, Standard Deviation

Journal: Stem Cells International

Article Title: Effects of Delta-Like Noncanonical Notch Ligand 1 Expression of Human Fetal Liver Hepatoblasts on Hematopoietic Progenitors

doi: 10.1155/2019/7916275

Figure Lengend Snippet: Flow cytometry analysis after Dlk1 + cell depletion. Flow cytometry analysis of Dlk1-negative human fetal liver-derived cells after five days in culture with Dlk1 + cells in direct contact (black bars), without Dlk1 + cells (white bars), or with Dlk1 + cells in inserts (grey bars). Data are given as means from n = 4 biological repeats ± standard deviation. ∗ indicates a statistically significant difference ( p ≤ 0.05). Abbreviations: fsc: forward scatter; ssc: side scatter; CD: cluster of differentiation.

Article Snippet: Antibodies used were mouse IgG1 and 2b lineage cocktail (Lin)-FITC, mouse IgG1 CD34-PE, mouse IgG1 CD38-AF700, mouse IgG1 CD31-BV421, mouse IgG1 CD45-BUV395 (all Becton Dickinson), mouse IgG1 CD326-PE (Miltenyi Biotec), and rabbit IgG Dlk1-AF647 (Bioss Antibodies, Woburn, MA); isotypes were mouse IgG1&2b-FITC, IgG1-PE, IgG1-AF700, IgG1-BV421, IgG1-BUV395 (all Becton Dickinson), and rabbit IgG-AF647 (Bioss Antibodies).

Techniques: Flow Cytometry, Derivative Assay, Standard Deviation

Journal: Stem Cells International

Article Title: Effects of Delta-Like Noncanonical Notch Ligand 1 Expression of Human Fetal Liver Hepatoblasts on Hematopoietic Progenitors

doi: 10.1155/2019/7916275

Figure Lengend Snippet: Colony forming unit assays after Dlk1 + cell depletion. CFU assays of Dlk1-negative human fetal liver-derived cells in five days preculture with Dlk1 + cells in direct contact (black bars), without Dlk1 + cells (white bars), or with Dlk1 + cells in inserts (grey bars). Data are given as means from n = 4 different repeats ± standard deviation. ∗ and ∗∗ indicate statistically significant differences ( p ≤ 0.05 and ∗∗ p ≤ 0.01, respectively). Abbreviations: CFU-E: colony forming unit-erythrocyte; BFU-E: burst forming unit-erythrocyte; CFU-GM: colony forming unit-granulocyte and macrophage; CFU-GEMM: colony forming unit-granulocyte, erythrocyte, macrophage, and megakaryocyte.

Article Snippet: Antibodies used were mouse IgG1 and 2b lineage cocktail (Lin)-FITC, mouse IgG1 CD34-PE, mouse IgG1 CD38-AF700, mouse IgG1 CD31-BV421, mouse IgG1 CD45-BUV395 (all Becton Dickinson), mouse IgG1 CD326-PE (Miltenyi Biotec), and rabbit IgG Dlk1-AF647 (Bioss Antibodies, Woburn, MA); isotypes were mouse IgG1&2b-FITC, IgG1-PE, IgG1-AF700, IgG1-BV421, IgG1-BUV395 (all Becton Dickinson), and rabbit IgG-AF647 (Bioss Antibodies).

Techniques: Derivative Assay, Standard Deviation

Journal: bioRxiv

Article Title: LINE-1 retrotransposon activation intrinsic to interneuron development

doi: 10.1101/2022.03.20.485017

Figure Lengend Snippet: a, Schematic of PV + and PV - /Tub + neuron isolation from pooled neonate (P0) litter hippocampal tissue. An anti-PV conjugated antibody (AF647) was used to label and isolate PV + cells. Freshly sorted PV - cells were subsequently labeled with an anti-Tub conjugated antibody (AF488) and sorted again to isolate PV - /Tub + neurons. b, Gating strategy and purity of fluorescence activated cell sorting (FACS) for PV + cells. c, As for (b), except showing PV - /Tub + neurons. d, Quality control qPCR of relative PV mRNA expression in sorted cells. PV mRNA enrichment in the PV + population was observed, as expected. Data are relative to GAPDH and presented as mean ± SD. * P =0.017, two-tailed t test, N (litters)=3.

Article Snippet: A rabbit anti-PV conjugated Alexa Fluor 647 antibody (Bioss bs-1299R-A647, dilution 1:2000) was directly added to the blocking buffer cell suspension and incubated for 1h at 4°C, then passed through a 40μm mesh cell strainer and subjected to flow cytometry.

Techniques: Isolation, Labeling, Fluorescence, FACS, Expressing, Two Tailed Test