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: Frontiers in Immunology

Article Title: NKG2D is a Key Receptor for Recognition of Bladder Cancer Cells by IL-2-Activated NK Cells and BCG Promotes NK Cell Activation

doi: 10.3389/fimmu.2015.00284

Figure Lengend Snippet: Cell surface expression of activating and inhibitory ligands by bladder cancer cells .

Article Snippet: Monoclonal antibodies specific for ULBP1, 2, 3, MICA, and MICB were purchased from R&D Systems (Abingdon, UK); ICAM-1/CD54 (Immunotech, Clone 84H10); Nectin 2/CD112 (Santa Cruz, Clone B-C12); CD155/PVR (Abcam, Clone D171); E-Cadherin (Immunotech, Clone 67A4); CD58/LFA-3 (Immunotech, Clone AICD58); CD106/VCAM-1 (Pharmingen, Clone 51-10C9); CD48 (Diaclone, Clone MEM102).

Techniques: Expressing