Journal: Yonago Acta Medica

Article Title: Prion Protein and Stage Specific Embryo Antigen 1 as Selection Markers to Enrich the Fraction of Murine Embryonic Stem Cell-Derived Cardiomyocytes

doi:

Figure Lengend Snippet: A: Flow cytometry analysis of cell surface PrP in EB derived from AB1 ES cells. Ordinate indicates the prevalence of DRAQ7-posisitve cells as dead cells and abscissa indicated the prevalence of PrP+ cells. The cells within a gated area show the population of living PrP+ cells.

Article Snippet: Dead cells were excluded with Draq7 (Biostatus, Shepshed, England).

Techniques: Flow Cytometry, Derivative Assay

Journal: bioRxiv

Article Title: Paired CRISPR screens identify mitochondrial metabolism and UBE2H as aneuploid-specific dependencies in human cancer cell lines

doi: 10.64898/2026.04.26.720636

Figure Lengend Snippet: Validation and mechanistic analysis of UBE2H A-D) Competition assay results showing fold dropout of sgRNAs targeting negative controls (ROSA26, AAVS1), positive controls (CDK9, CDC7), and UBE2H in A) SNU1 near-tetraploid control cells, B) SNU1 aneuploid subclone 114 (trisomy 9, 19), C) SNU1 aneuploid subclone 12 (trisomy 5, 10,17; pentasomy 2), D) SNU1 aneuploid subclone 24 (trisomy 1,8,10,14; DiY; pentasomy 5). Percent dropout is normalized relative to positive and negative controls. E) Colony formation following AZ3146 treatment (250 nM) relative to ethanol vehicle control in HCT116 wild-type cells, three UBE2H knockout clones, and UBE2H cDNA rescue clones. P-values from two-sided t-test relative to wild-type are shown. One sided t-test for overexpression clones rescue relative to knockout. F) Change in percentage of Annexin V and/or DRAQ7-positive cells following 96 h treatment with AZ3146 (1 µM), relative to ethanol-treated controls in HCT116 wild-type and UBE2H-KO clones. Two-sided t-tests. G) Diagram of sample preparation for Mass Spectrometry analysis. H-J) g:Profiler gene group enrichment analysis of mass spectrometry data. Protein abundance downregulated in HCT116 in H) AZ3146 treated versus wild-type, I) UBE2H-KO versus wild-type and J) AZ3146 treated UBE2H-KO versus AZ3146 treated wild-type cells. K) UBE2H RNA expression correlated to aneuploidy score in TCGA human tumor data. Pearson correlation and corresponding p-value. L) UBE2H RNA expression stratified by chromosome arm 7q copy number status; with 7q arm loss (-1), neutral (0) and gain (1) in TCGA data. Two-sided t-test p-values are shown. M) UBE2H RNA expression correlated to aneuploidy score for samples with a neutral copy of chromosome arm 7q in TCGA data. N) UBE2H RNA expression and aneuploidy score Pearson correlation coefficient and -log₂(p-value) per tumor types (TCGA). All significant correlations labeled with tumor abbreviation.

Article Snippet: Cells and media were harvested, resuspended in 100 μl annexin buffer and stained with AnnexinV FITC conjugate (Fisher Scientific, A13199) and then with 1 μl of DRAQ7 dead cell dye (Fisher Scientific, D15106).

Techniques: Biomarker Discovery, Competitive Binding Assay, Control, Knock-Out, Clone Assay, Over Expression, Sample Prep, Mass Spectrometry, Quantitative Proteomics, RNA Expression, Labeling

Journal: Journal of Neuroinflammation

Article Title: Variable sensitivity to complement-dependent cytotoxicity in murine models of neuromyelitis optica

doi: 10.1186/s12974-016-0767-4

Figure Lengend Snippet: Human complement (HC) is toxic to mouse neurons and oligodendrocytes in monocultures. IncuCyte live imaging of neurons, mature oligodendrocyte (OL), and oligodendrocyte progenitors (OPC) monocultures treated with HC (2% in neurons and 5% in OLs and OPCs) for 4 h. DRAQ7 was added in the culture medium to label dead cells. a , c , and e Phase contrast images show DRAQ7 staining ( red ) in the absence (CTRL) and presence (HC) of human complement. b , d , and f The percentage of DRAQ7+ neurons ( b ), OLs ( d ), and OPCs ( f ) increased rapidly following exposure to HC. g and h Astrocytes (AST) exposed to 5% HC for 4 h showed no evidence of cytotoxicity as measured and quantified by IncuCyte. CTRL: PBS. Scale bars 100 μm

Article Snippet: Each well was scanned with a 10× objective lens in nine randomly selected positions at 15 min intervals with high definition phase contrast and epifluorescence microscopy using the following filter sets: 460/524 nm (green fluorescence, to detect enhanced green fluorescent protein, eGFP) and 585/635 nm (red fluorescence, to detect the DRAQ7 dead cell nuclei; Cell Signaling Technology, Danvers, MA).

Techniques: Imaging, Staining

Journal: Journal of Neuroinflammation

Article Title: Variable sensitivity to complement-dependent cytotoxicity in murine models of neuromyelitis optica

doi: 10.1186/s12974-016-0767-4

Figure Lengend Snippet: Mixed cultures of neurons, oligodendrocytes, and astrocytes demonstrate reduced complement cytotoxicity. Neuroglial mixed cultures prepared from PLP-eGFP mouse pups were treated with 5% HC for 4 h in the presence of DRAQ7 and imaged using IncuCyte. a Phase contrast images of IncuCyte cultures treated with human complement (HC) at 0 and 4 h. Insert : Fluorescence image showing oligodendrocytes (PLP-eGFP; green ) and dead cells (DRAQ7; red ). Arrows : neurons. Arrow heads : astrocytes. b Quantitation of the percentage of DRAQ7+ neurons at 2 and 4 h in the presence of 2% or 5% HC. c Fluorescence image and quantitation of the percentage of PI+ dead neurons following exposure to 5% HC for 4 h. Left : fluorescence image of mixed cultures exposed to CTRL or HC and stained with PI ( red ) followed by NeuN ( green ) at 4 h. Note the dead neurons (PI+/NeuN+) in the HC-treated cultures. d , e IncuCyte fluorescence images ( left ) and quantitation of ( d ) differentiated oligodendrocyte (OL, arrows ) and ( e ) oligodendrocyte progenitor (OPC) death in mixed cultures. OPCs and differentiated OLs were defined by morphology. More than 150 differentiated oligodendrocytes (OL) were counted for each experiment. CTRL: PBS. Scale bars 100 μm

Article Snippet: Each well was scanned with a 10× objective lens in nine randomly selected positions at 15 min intervals with high definition phase contrast and epifluorescence microscopy using the following filter sets: 460/524 nm (green fluorescence, to detect enhanced green fluorescent protein, eGFP) and 585/635 nm (red fluorescence, to detect the DRAQ7 dead cell nuclei; Cell Signaling Technology, Danvers, MA).

Techniques: Fluorescence, Quantitation Assay, Staining

Journal: Journal of Neuroinflammation

Article Title: Variable sensitivity to complement-dependent cytotoxicity in murine models of neuromyelitis optica

doi: 10.1186/s12974-016-0767-4

Figure Lengend Snippet: Kinetics of neuron, OL, and OPC loss in the presence of NMO rAb #53-mediated astrocyte injury. a Fluorescence (left) and phase contrast IncuCyte (right) images of neuroglial mixed culture treated with rAb #53 plus 5% HC in the presence of DRAQ7 (PLP-eGFP oligodendrocytes [green] and DRAQ7 dead cells [red]). Arrows : neurons. Arrow heads : astrocytes. Circled area : focal region of astrocyte depletion. b , c , d , and e Quantitation of the percentage of DRAQ7+ AST ( b ), neuron ( c ), OL ( d ), and OPC ( e ) in the mixed cultures. Cells were identified as described in the . Statistical analyses were performed by multiple unpaired Student’s t test. ** p < 0.01, n > 3. Scale bars 50 μm

Article Snippet: Each well was scanned with a 10× objective lens in nine randomly selected positions at 15 min intervals with high definition phase contrast and epifluorescence microscopy using the following filter sets: 460/524 nm (green fluorescence, to detect enhanced green fluorescent protein, eGFP) and 585/635 nm (red fluorescence, to detect the DRAQ7 dead cell nuclei; Cell Signaling Technology, Danvers, MA).

Techniques: Fluorescence, Quantitation Assay