Journal: Infection and Immunity

Article Title: Leishmania-Infected Macrophages Are Targets of NK Cell-Derived Cytokines but Not of NK Cell Cytotoxicity

doi: 10.1128/iai.00079-11

Figure Lengend Snippet: FIG. 3. Leishmania infection does not alter the levels of NK cell regulatory molecules on the surface of macrophages or DCs. BM-M, PE-M, or BM-DCs were infected with L. infantum promastigotes at a parasite/cell ratio of 7:1 for 14 h. CD11b F4/80 M and CD11c CD11b DCs were stained for the expression of the NK cell-receptor ligands MHC class I and Qa-1b (A; inhibitory), CD48 (B; inhibitory or activating), or Rae-1 and MULT-1 (C; activating). The histograms represent the expression levels of these molecules on the surface of uninfected (solid lines) or infected (dashed lines) cells. Isotype controls (shaded) were included for each staining. The data are representative of 4 independent experiments.

Article Snippet: For surface phenotyping and cell sorting, fluorochrome (fluorescein isothiocyanate, phycoerythrin, or allophycocyanin [APC])-labeled or biotinylated monoclonal antibodies against the following mouse antigens were used: NK1.1 (PK136), CD3ε (145-2C11), CD11b (M1/70), CD11c (HL3), CD48 (HM48-1), and mouse UL-16-binding protein-like transcript 1 (MULT-1; 5D10) (all from eBioscience); Qa-1 (6A8.6F10.1A6) and H-2kb (AF6-88.5; BD Biosciences, Heidelberg, Germany); F4/80 (CI:A3-1; AbD Serotec, Düsseldorf, Germany); and pan-retinoic acid early transcript 1 (panRae-1) (186107; R&D Systems, Wiesbaden, Germany).

Techniques: Infection, Staining, Expressing

Journal: Cell Death & Disease

Article Title: Acute lymphoblastic leukemia-derived extracellular vesicles affect quiescence of hematopoietic stem and progenitor cells

doi: 10.1038/s41419-022-04761-5

Figure Lengend Snippet: A Procedure followed to identify populations of murine cells that take in fluorescent ALL EVs. B Flow cytometry on total BM cells demonstrating that primitive hematopoietic cells (Sca1 + mCD45 + ) take in fluorescent T-ALL and B-ALL EVs (ATTO488 EVs). C Flow cytometry gating strategy used to analyze further subpopulations among the primitive Lin − cells and their capacity to take in ALL EVs. D Mean fluorescence intensity (MFI) on Lin − cells showing that HSPC (LSK; Lin − Sca1 + c-Kit + ) and HSC (SLAM; LSK CD150 + CD48 − ) can take in ALL EVs (ATTO488 EVs), while LK (Lin − c-Kit + Sca1 − ) cells and the c-Kit negative (c-Kit − ) cells cannot. The intake of SLAM cells was higher than LSK cells. Data are compared to c-Kit − cells and are shown as means ± SD; n = 3 mice. P value measured by one-way Anova with Tukey’s multiple comparison test; $ P < 0.0001. E Among Lin − cells, cells showing intake of ALL EVs (ATTO488 EVs positive cells; pos) and no intake (ATTO488 EVs negative cells; neg). To examine the cell intake of EVs, Lin − cells are treated with HSP70-ATTO488 EVs, for 30 min to 2 h. Data showing that ALL EVs have the potential to rapidly bind, within 30 min, ~4% of the Lin − cells. Data are shown as means ± SD; n = 4 biological replicates. P value measured by one-way Anova with Tukey’s multiple comparison test; **** P < 0.0001. F CFU assay is assessed for 30,000 pos and neg cells purified by FACS, after 7 days on semi-solid media. Data showing that pos cells are more primitive, while they produce more CFU-GM colonies when compared with neg cells. Quantity of CFU (left panel) and distribution (right panel). Data are shown as means ± SD; n = 3 mice. P value measured by two-tailed unpaired Student’s t test; **** P < 0.0001; ns, non-significant.

Article Snippet: We used the following antibodies; Sca1-APC-Cy7 (1:100, 108126, Biolegend, Paris, France), Sca1-VB-FITC (1:100, 130-116-490, Miltenyi Biotec), CD48-PE-Cy7 (1:100, 560731, BD Biosciences, Le Pont de Claix, France), CD48-FITC (1:100, 103404, Biolegend), CD48-APC (1:100, 130-124-727, Miltenyi Biotec), c-Kit-BV650 (1:100, 135125, Biolegend), c-Kit-PE-Cy7 (1:100, 558163, BD Biosciences), CD150-PerCP-Cy5.5 (1:100, 115921, Biolegend), murine CD45.2-Vioblue (1:100, 130-102-980, Miltenyi Biotec).

Techniques: Flow Cytometry, Fluorescence, Comparison, Colony-forming Unit Assay, Purification, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Acute lymphoblastic leukemia-derived extracellular vesicles affect quiescence of hematopoietic stem and progenitor cells

doi: 10.1038/s41419-022-04761-5

Figure Lengend Snippet: A NSG mice were injected with fluorescent EVs (ATTO488 EVs) by i.v. injections at 10 10 particles/mouse. Mice were sacrificed 24 h after the injection, and florescence was determined by flow cytometry on Lin − , LSK (c-Kit + Sca1 + ) and SLAM (LSK CD150 + CD48 − ) cells. B Procedure followed to treat NSG mice with ALL EVs or control EVs, through three consecutive i.v. injections, at 10 10 particles/mouse/every 10 days. Ten days after the third injection, mice were sacrificed to perform experiments on Sca1 + cells. C The absolute number of Sca1 + cells detected in BM. Data are shown as means ± SD; n = 4 mice. D The number of hematopoietic progenitors (CD45 + Sca1 + c-Kit + cells) and HSC (SLAM; LSK CD150 + CD48 − cells) were determined by flow cytometry. Data are shown as means ± SD; n = 4 mice. E Sca1 + cells (10 5 cells) isolated from mice were analyzed on semi-solid methylcellulose media for growth of murine hematopoietic CFU. Data show the percentage of total CFU and CFU-GM per Sca1 + cells, as well as the distribution among colonies. Data are shown as means ± SD; n = 4 mice. F Sca1 + cells (3 × 10 5 cells) were injected in C57BL/6.SJL (Ly.1) mice and the hematopoietic reconstitution was examined by flow cytometry, 4 weeks after the transplantation. Absolute number of CD45.2 total and Lin − BM cells. Data are shown as means ± SD; n = 3 transplanted mice. G Hematopoietic reconstitution for progenitors (LSK cells) and HSC (SLAM cells). Absolute number of CD45.2 cells in BM. Data are shown as means ± SD; n = 3 transplanted mice. On this figure, P value is measured by one-way Anova with Tukey’s multiple comparison test; ** P < 0.01; *** P < 0.001; ns, non-significant.

Article Snippet: We used the following antibodies; Sca1-APC-Cy7 (1:100, 108126, Biolegend, Paris, France), Sca1-VB-FITC (1:100, 130-116-490, Miltenyi Biotec), CD48-PE-Cy7 (1:100, 560731, BD Biosciences, Le Pont de Claix, France), CD48-FITC (1:100, 103404, Biolegend), CD48-APC (1:100, 130-124-727, Miltenyi Biotec), c-Kit-BV650 (1:100, 135125, Biolegend), c-Kit-PE-Cy7 (1:100, 558163, BD Biosciences), CD150-PerCP-Cy5.5 (1:100, 115921, Biolegend), murine CD45.2-Vioblue (1:100, 130-102-980, Miltenyi Biotec).

Techniques: Injection, Flow Cytometry, Control, Isolation, Transplantation Assay, Comparison

Journal: Cell Death & Disease

Article Title: Acute lymphoblastic leukemia-derived extracellular vesicles affect quiescence of hematopoietic stem and progenitor cells

doi: 10.1038/s41419-022-04761-5

Figure Lengend Snippet: Mice were injected with 5 × 10 5 cells (T-ALL) or 10 5 cells (B-ALL) and murine Sca1 + cells were recovered when mice were developing ALL disease (day 35). A The number of Sca1 + cells recovered from the BM of control NSG mice and PDX mice developing T-ALL or B-ALL. Data are shown as means ± SD; n = 5 mice. B Analysis of Sca1 + cells by flow cytometry showing loss of hematopoietic progenitors (CD45 + c-Kit + ) and HSC (SLAM; CD150 + CD48 − ) in T-ALL and B-ALL PDX mice. Data are shown as means ± SD; n = 5 mice. C 25% of the Sca1 + cells were cultured in methylcellulose media and hematopoietic CFU were observed at day 7; scale bar represents 5 mm. Data are shown as means ± SD; n = 3 mice. D 75% of the Sca1 + cells were cultured in media for growth of mesenchymal CFU for 10 days, scale bar represents 1 cm. Data are shown as means ± SD; n = 3 mice. On this figure, P value is measured by one-way Anova with Tukey’s multiple comparison test; *** P < 0.001; **** P < 0.0001; ns non-significant.

Article Snippet: We used the following antibodies; Sca1-APC-Cy7 (1:100, 108126, Biolegend, Paris, France), Sca1-VB-FITC (1:100, 130-116-490, Miltenyi Biotec), CD48-PE-Cy7 (1:100, 560731, BD Biosciences, Le Pont de Claix, France), CD48-FITC (1:100, 103404, Biolegend), CD48-APC (1:100, 130-124-727, Miltenyi Biotec), c-Kit-BV650 (1:100, 135125, Biolegend), c-Kit-PE-Cy7 (1:100, 558163, BD Biosciences), CD150-PerCP-Cy5.5 (1:100, 115921, Biolegend), murine CD45.2-Vioblue (1:100, 130-102-980, Miltenyi Biotec).

Techniques: Injection, Control, Flow Cytometry, Cell Culture, Comparison

Journal: Genes

Article Title: Novel PIGT Variant in Two Brothers: Expansion of the Multiple Congenital Anomalies-Hypotonia Seizures Syndrome 3 Phenotype

doi: 10.3390/genes7120108

Figure Lengend Snippet: ( A ) Abundance of glycosylphosphatidylinositol (GPI)-anchored proteins measured by flow cytometry in two controls, in patient 1 in peripheral blood, and in patient 1 and 2 in skin fibroblasts ( B ). Cells were stained with fluorochrome conjugated aerolysin (FLAER) to detect total GPI-anchors and with fluorochrome conjugated antibodies to detect GPI-anchored proteins (CD14, CD16, CD24, CD48, CD49, and CD90). The y -axis displays Median Fluorescent Intensities (MFI) of proteins on indicated cell populations.

Article Snippet: Blood cells from patient 1 were stained with the following lineage markers and antibodies: CD19 (clone HIB19), and CD45 (Clone HI30) (both purchased from BD Bioscience, San Jose, CA, USA) as well as antibodies against the following GPI-APs: CD14 (M5E2), CD16 (3G8), CD24 (ML5), CD48 (MEM-102), and fluorochrome conjugated aerolysin (FLAER) (Cedarlane, Burlington, NC, USA), which specifically binds GPI-anchors.

Techniques: Flow Cytometry, Staining

Journal: Scientific Reports

Article Title: Macrophages lift off surface-bound bacteria using a filopodium-lamellipodium hook-and-shovel mechanism

doi: 10.1038/srep02884

Figure Lengend Snippet: Deconvolved confocal fluorescence and interference reflection micrographs (IRM). Samples were stained for macrophage actin (white), CD48 (yellow) and E. coli (red). CD48 exclusively localized within the macrophage membrane including filopodia (see arrowheads and x-z and y-z cross sections). Filopodia (FP) and lamellipodia (LP) – bacteria contacts were suggested by IRM.

Article Snippet: 35 mm tissue culture dishes (Corning CellBIND, 3294) were incubated for 75 min at 37°C with either 100 μg/ml mono-mannose-BSA (Dextra Labs, NGP1108), 20 μg/ml RNaseB (Sigma Aldrich, R1153) or 20 μg/ml recombinant mouse CD48 (R&D Systems, 3327-CD) in 0.02 M bicarbonate buffer.

Techniques: Fluorescence, Staining, Membrane, Bacteria

Journal: Scientific Reports

Article Title: Macrophages lift off surface-bound bacteria using a filopodium-lamellipodium hook-and-shovel mechanism

doi: 10.1038/srep02884

Figure Lengend Snippet: (a) Addition of 2% soluble α−D-mannopyranoside inhibitor (αMM) and CD48 antibodies substantially reduced the rate of E. coli phagocytosis. Uptake rates were analysed for 10 independent macrophages during 10 min live cell experiments and normalized by the time-averaged number of bacteria available in the zone explored by filopodia. Mean values are given as horizontal line. (b) Bacterial uptake was observed at all sides of the macrophages and was independent of the direction of fluid flow (0.1 ml/min flow rate/0.06 pN/μm 2 shear stress). (c) Filopodia length distribution as analysed from fixed macrophages. 95% of the filopodia (n = 400) have a length less than 8 μm, which we defined here as the filopodia sensing zone (yellow area).

Article Snippet: 35 mm tissue culture dishes (Corning CellBIND, 3294) were incubated for 75 min at 37°C with either 100 μg/ml mono-mannose-BSA (Dextra Labs, NGP1108), 20 μg/ml RNaseB (Sigma Aldrich, R1153) or 20 μg/ml recombinant mouse CD48 (R&D Systems, 3327-CD) in 0.02 M bicarbonate buffer.

Techniques: Bacteria, Shear

Journal: Scientific Reports

Article Title: Macrophages lift off surface-bound bacteria using a filopodium-lamellipodium hook-and-shovel mechanism

doi: 10.1038/srep02884

Figure Lengend Snippet: (a) Accumulation of type I fimbriated E. coli FimH-j96 bacteria on flow chamber bottom glass surfaces coated with CD48, mono-mannose bovine serum albumin (1 M) and tri-mannose RNaseB (3 M) under varying shear stresses τ (1 pN μm −2 = 1 Pascal or 10 dynes cm −2 ). E. coli accumulation was analysed after 5 min. As negative controls, either 2% of a mono-mannose inhibitor (αMM) was added to the media ( ), or the non-fimbriated E. coli parent strain AAEC191A was used (Δfim, ). (b) Fraction of E. coli FimH-j96 that adhered firmly on CD48, 1 M and 3 M. Bacteria were defined firmly adhering if they moved less than one-half of a bacterial diameter over >30 s. (c) Effect of different FimH variants on bacterial accumulation to 1 M and CD48. While the low binding FimH-f18 strain only adhered to CD48 and 1 M above a critical shear stress, FimH-j96 E. coli accumulated on CD48 without any shear threshold.

Article Snippet: 35 mm tissue culture dishes (Corning CellBIND, 3294) were incubated for 75 min at 37°C with either 100 μg/ml mono-mannose-BSA (Dextra Labs, NGP1108), 20 μg/ml RNaseB (Sigma Aldrich, R1153) or 20 μg/ml recombinant mouse CD48 (R&D Systems, 3327-CD) in 0.02 M bicarbonate buffer.

Techniques: Bacteria, Shear, Binding Assay

Journal: Scientific Reports

Article Title: Macrophages lift off surface-bound bacteria using a filopodium-lamellipodium hook-and-shovel mechanism

doi: 10.1038/srep02884

Figure Lengend Snippet: (a) The mannosylated membrane anchored surface receptor CD48 of macrophages specifically binds to the bacterial fimbrial tip adhesin FimH, which contains a single mannose-binding pocket in the lectin domain (Hook). (b) Due to filopodia retractions, the bacterial fimbriae are elongated. As bacteria adhere tightly to substrate surfaces via multiple bonds, the macrophages fail to pull bacteria off the surface via the hook alone. (c) To facilitate uptake, macrophages protrude lamellipodia towards the bacterium to sequentially break the bonds that anchor E. coli to the surface (Shovel). (d) Once the bacterium is completely lifted off the substrate and lies on the lamellipodium, a phagocytic cup is formed to internalize the bacterium.

Article Snippet: 35 mm tissue culture dishes (Corning CellBIND, 3294) were incubated for 75 min at 37°C with either 100 μg/ml mono-mannose-BSA (Dextra Labs, NGP1108), 20 μg/ml RNaseB (Sigma Aldrich, R1153) or 20 μg/ml recombinant mouse CD48 (R&D Systems, 3327-CD) in 0.02 M bicarbonate buffer.

Techniques: Membrane, Binding Assay, Bacteria