high content immunofluorescence imaging analysis system  (Danaher Inc)

Journal: The Journal of Clinical Investigation

Article Title: CRISPR/Cas9 screens identify LIG1 as a sensitizer of PARP inhibitors in castration-resistant prostate cancer

doi: 10.1172/JCI179393

Figure Lengend Snippet: ( A ) Immunoblot of PARP1, PAR, and H3 (used as loading control) in the chromatin-bound extract (CBE) of 22Rv1 transduced with the indicated sgRNAs and treated for 3 days with DMSO, as control, or OLA. ( B ) Comet tail moment measured by alkaline comet assay in 22Rv1 transduced with the indicated sgRNAs and treated as in A . The orange dots and bars indicate the mean value of each replicate and the mean ± SEM of the 3 experiments, respectively. P values were determined using 1-way ANOVA and Tukey’s multiple-comparisons test. Images are representative of the comet assays. Scale bars: 100 μm. ( C ) Quantification of cells with 5 or more γH2AX foci measured by immunofluorescence. Data are presented as mean ( n = 2 biological replicates). Images are representative of γH2AX (green) and nucleus (Hoechst, blue) immunostaining in 22Rv1 transduced with the indicated sgRNAs and treated as in A . Scale bars: 50 μm. ( D ) Immunoblot of LIG1, p-ATM, ATM, VINC (used as loading control), p-CHK1, CHK1, ACTB (used as loading control), γH2AX, and TUBB (used as loading control) in 22Rv1 transduced with the indicated sgRNAs and treated as in A . ( E ) Percentage of cells with 10 or more γH2AX foci measured by immunofluorescence after OLA washout. 22Rv1 transduced with the indicated sgRNAs were treated with 1 μM OLA for 3 days and then grown without treatment for 0, 1, 8 or 24 hours. Data are presented as mean ± SEM ( n = 3 biological replicates). P values were determined using 2-way ANOVA and Bonferroni’s multiple-comparisons test on control (sgNTC and sg EGFP ) and sg LIG1 samples. * P ≤ 0.05; ** P ≤ 0.01.

Article Snippet: Immunofluorescence analysis was performed with ImageXpress Micro Confocal (Molecular Devices).

Techniques: Western Blot, Control, Transduction, Alkaline Single Cell Gel Electrophoresis, Immunofluorescence, Immunostaining

Journal: The Journal of Clinical Investigation

Article Title: CRISPR/Cas9 screens identify LIG1 as a sensitizer of PARP inhibitors in castration-resistant prostate cancer

doi: 10.1172/JCI179393

Figure Lengend Snippet: ( A ) Schematic diagram of the in vivo experiments. ( B ) Immunoblot analysis of LIG1 and ACTB (used as loading control) in 6 exemplary xenograft tumor samples collected after the in vivo experiments described in A . ( C ) Scatter plots of the tumor volume measured during treatment with vehicle or AZD5305. Data are presented as mean + SD. ( D ) Percentage of cells with 5 or more γH2AX foci measured by immunofluorescence in FFPE xenograft tumor samples collected after the in vivo experiments described in A . Images are representative of 3 FFPE xenografts tumor sections stained for γH2AX (red) and nucleus (DAPI, blue). Scale bars: 12 μm. Data are presented as mean ± SD. P values were determined using 2-tailed unpaired t test. * P ≤ 0.05; ** P ≤ 0.01; **** P ≤ 0.0001.

Article Snippet: Immunofluorescence analysis was performed with ImageXpress Micro Confocal (Molecular Devices).

Techniques: In Vivo, Western Blot, Control, Immunofluorescence, Staining

Journal: iScience

Article Title: Uncovering specificity of endogenous TAU aggregation in a human iPSC-neuron TAU seeding model

doi: 10.1016/j.isci.2021.103658

Figure Lengend Snippet: Standardization of a robust platform for hiPSC conversion to cortical neurons (A) Representative phase-contrast images showing the expected morphology of hiPSCs throughout differentiation into hiPSC-neurons. i: hiPSC before harvesting; ii: day 9; iii: day 15; iv: day 25; v: day 28/DIV 3; vi: day 46/DIV 21. DIV indicates day in vitro of neuronal maturation. (B) Day 10 checkpoint comparing the immunofluorescence (IF) staining ( left ) and quantification ( right ) of “pass” (PAX6 >80%; SOX10 < 1%, AP2α <1%) and “fail” batches. Graph represents mean +SD ( n = 3 replicate wells, 25 fields per well). (C) Representative IF images and quantification reveal a reduction in SOX2+ proliferative neural progenitor cells and total cell number following treatment with the γ-secretase inhibitors (GSI) DAPT or RO4929097, resulting in a more homogeneous neuronal (MAP2/TUBB3+) population. Graphs represent mean +SD ( n = 3 replicate wells, 30 fields per well). ∗∗∗∗p < 0.0001 according to one-way ANOVA with Dunnett's test. (D) Plating conditions were optimized to reduce cell toxicity, prevent clumping, and achieve an evenly distributed MAP2+/TUBB3+ neurite network. Optimal conditions are described in the STAR methods section. (E) DIV21 hiPSC-neurons express pan-neuronal markers including TUBB3, MAP2, and CP27 (human total tau) as well as markers consistent with a telencephalic (FOXG1), deep layer (TBR1) cortical identity. (F) Heatmap with unbiased hierarchical clustering depicts a subset of differentially expressed genes at the hiPSC, hiPSC-NPC, and hiPSC-neuron stages, demonstrating consistency and reproducibility in producing a cortical excitatory fate. Relative expression values are scaled per gene to indicate the cell stage with the highest expression. Scale bars: 100 μm. See also Figure S1 .

Article Snippet: High content immunofluorescence imaging was conducted on either the Operetta or Opera Phenix instrument (PerkinElmer) using Harmony (PerkinElmer) for image acquisition and analysis.

Techniques: In Vitro, Immunofluorescence, Staining, Expressing

Journal: iScience

Article Title: Uncovering specificity of endogenous TAU aggregation in a human iPSC-neuron TAU seeding model

doi: 10.1016/j.isci.2021.103658

Figure Lengend Snippet: Endogenous tau aggregates in hiPSC-neurons display characteristic features of pathological tau (A) Schematic diagram of tau protein domains and antibody-binding sites for MC1, AT8, PHF1, and pS422. (B–K) Representative IF images showing immunoreactivity of endogenous tau aggregates in MAPT -WT hiPSC-neurons seeded with 0.5 nM SI-AD and in MAPT -P301S/E10+14/E10+16 hiPSC-neurons seeded with 2N4R P301L sPHF for MC1 (B, C, G, H), AT8 (D, I), PHF1 (E, J), and pS422 (F, K). (L) Biochemical analysis using homogeneous time resolved fluorescence (HTRF) and a monoclonal tau antibody that specifically recognizes tau aggregates found in AD brain lysate, brain-derived (SI-AD) and recombinant (2N4R P301L sPHF) tau seeds. HTRF detects tau aggregates found in conditions that produced detectable aggregation by MC1 immunofluorescence but not in controls, which display a signal similar to background (PBS) or control brain lysate. Graph represents mean +SD ( n = 2 technical replicates). Scale bar: 10 μm.

Article Snippet: High content immunofluorescence imaging was conducted on either the Operetta or Opera Phenix instrument (PerkinElmer) using Harmony (PerkinElmer) for image acquisition and analysis.

Techniques: Binding Assay, Fluorescence, Derivative Assay, Recombinant, Produced, Immunofluorescence, Control