Journal: Cancer cell

Article Title: Synthetic Lethal and Convergent Biological Effects of Cancer-Associated Spliceosomal Gene Mutations

doi: 10.1016/j.ccell.2018.07.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Five days after injection, BM cells were harvested from the legs (femora and tibiae) and hip bones, and lineage-depletion was performed with biotin-conjugated antibodies against B220 (RA3-6B2), CD19 (1D3), CD3 (17A2), CD4 (GK1.5), CD8a (53-6.7), CD11b (M1/70), Gr-1 (RB6-8C5), NK1.1 (PK136) and Ter119, labeled with anti-biotin MicroBeads (130-090-485; Miltenyi Biotec), and lineage-negative (Lin − ) cells were magnetically separated using MACS columns according to the manufacturer’s instructions.

Techniques: Recombinant, Reporter Assay, Transgenic Assay, Plasmid Preparation, Software

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet: TL1A Enhances the Activation of MAIT Cells Suboptimally Stimulated with IL-12 and IL-18 CD8 + T cells were enriched from healthy peripheral blood mononuclear cells (PBMCs) and stimulated overnight with different combinations of cytokines: IL-12 at 2 ng/mL, IL-18 at 50 ng/mL, IL-15 at 25 ng/mL, and TL1A from 0.01 to 100 ng/mL as indicated. (A–C) Proportions of CD8 + MAIT/CD161 + or CD161 − cells producing IFN-γ (A), TNF-α (B), or CD69 (C) following overnight stimulation with suboptimal concentrations of IL-12 and IL-18, plus varying concentrations of TL1A. (D) Representative histograms showing the expression of IFN-γ, TNF-α, GrB, and CD69 by MAIT cells after stimulation with different combinations of cytokines. (E–H) Frequency of MAIT cells expressing IFN-γ (E), TNF-α (F), GrB (G), and CD69 (H) upon stimulation with the indicated cytokines. Data were acquired from seven donors in 2–3 experiments. Error bars represent means ± SEM. Differences among conditions were analyzed by Friedman tests with Dunn’s multiple comparison tests. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also Figure S1 .

Article Snippet: Anti-human CD161 (clone 191B8) APC , Miltenyi , Cat# 130-113-590; RRID: AB_2733346.

Techniques: Activation Assay, Expressing, Comparison

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet: TCR-Mediated Activation of MAIT Cells Leads to the Expression of Tissue-Repair-Associated Molecules and Accelerates Wound Healing (A–C) Gene set enrichment summary plots for stimulated sorted MAIT cell-versus-unstimulated cell-ranked genes. Depicted are the individual plots for TCR-stimulated versus UT in (A), TC-stimulated versus UT in (B), and C versus unstimulated in (C). Non-significant for C versus UT, normalized enrichment score (NES) = 1.63; p < 0.0002 for TCR versus UT, NES = 1.57; and p < 0.0002 for TC versus UT. Data were acquired from three donors in one experiment. (D) Flow cytometry analysis of the expression of TNF-α, furin, and CCL3 by CD161 ++ /MAIT CD8 + T cells in response to fixed E. coli presented by THP1 cells in the presence or absence of an anti-MR1 (αMR1) blocking antibody at the 72-h time point. (E) Statistical analysis of the expression of the effector molecules shown in (D). (F) Caco2 cells were grown to confluency and scratched with a WoundMaker device to perform in vitro wound-healing assays. Cells were supplemented with different supernatants collected from 72-h cocultures of enriched CD8 T cells with E. coli -loaded THP1 cells in the presence or absence of αMR1, as indicated. The open wound areas were quantified as percentages of the initial wound size in the Caco2 cultures. Data points are mean ± SEM and were acquired from five biological replicates in two experiments. (G) Representative pictures of the closure of the wounds in Caco2 cultures treated as in (F) were assessed with time-lapse imaging over a time course of 36 h. Data were acquired from seven donors in three experiments. Differences among conditions were analyzed by two-way ANOVA. ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.001. Scale bars, 250 μm. See also Figure S5 and .

Article Snippet: Anti-human CD161 (clone 191B8) APC , Miltenyi , Cat# 130-113-590; RRID: AB_2733346.

Techniques: Activation Assay, Expressing, Flow Cytometry, Blocking Assay, In Vitro, Imaging

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet: MAIT Cells Can Be Found Close to and within the Colonic Epithelium (A–G) Representative images showing the expression of Va7.2, CD161, CD8, PLZF, CD3, and CD103 in the lamina propria and the epithelium of fixed samples of colonic polyp tissue. Samples were mounted on cytometer chips and iteratively stained with sets of three directly fluorochrome-conjugated antibodies as described in the methods section. Depicted are a merged picture (A) and all the individual stains for Va7.2 (B), CD161(C), CD8 (D), PLZF (E), CD3 (F), and CD103 (G). White arrows mark cells showing co-expression of Va7.2, CD161, PLZF, and CD3 that were defined as MAIT cells here. Note that while CD8 was co-expressed in most of them, CD8− MAITs (arrow + asterisk) could also be found. In contrast, CD103 was rarely co-expressed on MAITs (arrow + diamond). During the iterative staining process dust particles and other detritus can be picked up by the solution flowing over the tissue creating autofluorescent artifacts (1–4). While some of these get washed away after completion of the staining cycle (1, 4), others present during multiple imaging rounds (2, 3). Scale bars, 50μm.

Article Snippet: Anti-human CD161 (clone 191B8) APC , Miltenyi , Cat# 130-113-590; RRID: AB_2733346.

Techniques: Expressing, Cytometry, Staining, Imaging

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet:

Article Snippet: Anti-human CD161 (clone 191B8) APC , Miltenyi , Cat# 130-113-590; RRID: AB_2733346.

Techniques: Virus, Recombinant, Reverse Transcription, Activation Assay, Staining, Software

Journal: Scientific Reports

Article Title: Cell release during perfusion reflects cold ischemic injury in rat livers

doi: 10.1038/s41598-020-57589-4

Figure Lengend Snippet: Alterations in liver-resident immune cell release into the perfusate after cold ischemia. ( a ) Percentage of presumed Kupffer cells (left/orange), liver-resident natural killer cells (also known as pit cells) (middle/red), and dendritic cells (right/yellow) in the perfusate, relative to the total number of nucleated cells (TNCs) that are released into the perfusate from fresh (n = 4), 24-h-cold ischemic (CI) (n = 5), and 72-h-CI (n = 4) livers. The perfusate recirculated during 3 hours of subnormothermic machine perfusion. Stars denote statistical significance (two-way ANOVA, followed by Tukey’s post-hoc test): *0.01 < p < 0.05; **0.001 < p < 0.01; ***0.0001 < p < 0.001; ****p < 0.0001. Error bars: SEM. ( b ) Imaging flow cytometry for the quantification of presumed Kupffer cells (CD45+/CD105−/SE1−/CD14+) and pit cells (CD45+/NKRP1A+/CD3−/OX62−). Dendritic cells were manually selected from a CD45+/NKRP1A−/CD3−/OX62+ population (not shown). Left: fresh livers. Right: 24-h-CI livers. ( c ) Representative images of surface marker expression images of Kupffer cells (top), pit cells (middle), and dendritic cells (below). Scale bars: 5 µm.

Article Snippet: The aliquot for panel 3 was additionally stained for the detection of pit p cells and dendritic p cells with Alexa 405-conjugated NKRP1A (1:80; Novus Biologicals; Cat# NB100-65297AF405), FITC-conjugated CD3 (1:50; Invitrogen, Carlsbad, CA, USA; Cat# 11-0030-82), and PE-conjugated OX62 (1:100; Thermo Fisher; Cat# 12-1030-82) antibodies.

Techniques: Imaging, Flow Cytometry, Marker, Expressing

Journal: ESC Heart Failure

Article Title: Metformin inhibits proliferation of residing fibroadipogenic progenitor cells from failing human hearts

doi: 10.1093/eschf/xvag001

Figure Lengend Snippet: CD45 − CD31 − CD34 + cells isolated from the human myocardium possess fibro- and adipogenic differentiation potential. For subsequent experiments, mononuclear cells were isolated from the left ventricle of human hearts from heart transplantation recipients. CD45 − CD31 − CD34 + cells were further selected by fluorescence-activated cell sorting for in vitro experimentation. ( A ) PDGFRA is a marker of mesenchymal stem cells (multipotent stem cells) and was exclusively expressed in one cluster with a fibroblast phenotype. CD34 was expressed in the same cluster as PDGFRα , as well as in a large cluster of PECAM1 (CD31) positive cells, which represent endothelial cells. PTPRC (CD45) was exclusively expressed in two clusters representing macrophages and T/NK cells. ( B ) Live CD45 − CD31 − CD34 + cells isolated by fluorescence-activated cell sorting. ( C ) Isolated CD45 − CD31 − CD34 + cells can be grown in cell culture and express PDGFRα assessed by immunofluorescence staining (magnification 10X). ( D and E ) Isolated CD45 − CD31 − CD34 + cells increases COL-1 expression when grown in fibrogenic media, suggesting that they differentiate into fibroblasts. ( F and G ) Isolated CD45 − CD31 − CD34 + cells start to express PLIN-1 when grown in adipogenic media, suggesting that they differentiate into adipocytes. Thus, these cells may represent human cFAPs. The image magnifiation for D-G is 20X

Article Snippet: Anti CD45 , Miltenyi , Cat. no. 130-114-123.

Techniques: Isolation, Transplantation Assay, Fluorescence, FACS, In Vitro, Marker, Cell Culture, Immunofluorescence, Staining, Expressing

Journal: ESC Heart Failure

Article Title: Metformin inhibits proliferation of residing fibroadipogenic progenitor cells from failing human hearts

doi: 10.1093/eschf/xvag001

Figure Lengend Snippet: Metformin in pharmacological doses (0.1 mM) inhibits proliferation of fibrogenic progenitors. Mononuclear non-muscle cells were isolated from the left ventricle of explanted human hearts from heart transplantation recipients. Subsequently, CD45 − CD31 − CD34 + cells were isolated by fluorescence-activated cell sorting for in vitro experimentation. ( A and B ) COL-1 and COL-6 mRNA expression in cFAPs grown in fibrogenic media and incubated with metformin (0.1 mM) and cimetidine (0.1 mM) alone and in combination. ( C ) Quantification of proliferation assay as per cent 5-ethynyl-2'-deoxyuridine (EDU) positive cells. Individual donors ( A–C ) are indicated by the shape of the dots. ( D ) Cell proliferation assays (EDU) and apoptosis assays (TUNEL) performed on cFAPs in cell culture incubated with metformin (0.1 mM) and cimetidine (0.1 mM) alone and in combination (magnification 10X) (* P < .05)

Article Snippet: Anti CD45 , Miltenyi , Cat. no. 130-114-123.

Techniques: Isolation, Transplantation Assay, Fluorescence, FACS, In Vitro, Expressing, Incubation, Proliferation Assay, TUNEL Assay, Cell Culture