dhl 60 cells  (Hitachi Ltd)

Journal: bioRxiv

Article Title: Benchmarking three simple DNA staining-based image metrics for live-cell tracking of chromatin organization

doi: 10.64898/2026.03.30.715467

Figure Lengend Snippet: (A, B) Representative fluorescence images of dHL-60 cell nuclei at early chromatin reorganization (compact; blue box, A) and late chromatin reorganization (decompact; orange box, B ) stages during NETosis, stained with SPY650-DNA. Raw pixel values are shown with corresponding intensity colorbars. Yellow dashed lines indicate nuclear ROI boundaries. Scale bars, 5 µm. These two chromatin states serve as the basis for metric illustration in panels C– H . (C, D) CV (coefficient of variation; σ/μ) computed from the raw pixel intensity distribution within the nuclear ROI of the compact (C) and decompact (D) nuclei in (A, B) . Histograms show the distribution of raw pixel intensities for each nucleus; vertical lines indicate the mean (dashed) and ±1 SD (dotted). CV decreases from the compact (CV = 0.583) to the decompact state (CV = 0.326), reflecting narrowing of the intensity distribution. (E, F) 1-Gini (= B/(A+B)) computed from the Lorenz curve of the min-max normalized pixel distribution for the compact (E) and decompact (F) nuclei. Left in each panel: conceptual illustration showing how 1-Gini reflects signal uniformity across spatial units — when equal total signal is concentrated in a few spatial units (1-Gini = 0.25) versus distributed across many units (1-Gini = 0.67), 1-Gini increases proportionally. Disk area is proportional to pixel intensity. Right in each panel: Lorenz curves from the example nuclei in (A, B) . The diagonal dashed line represents perfect signal uniformity; area A (gray) represents the region between the diagonal and the Lorenz curve, and area B (condition color) the area below the curve. 1-Gini increases from the compact (1-Gini = 0.620) to the decompact state (1-Gini = 0.797), reflecting greater signal homogeneity. (G, H) DSI (Diffuse Signal Index; fraction of I norm > τ) computed as the fraction of min-max normalized pixels exceeding a threshold τ = 0.3 for the compact (G) and decompact (H) nuclei. For each condition, the normalized intensity ( I norm ) map is shown alongside a 3D surface plot of I norm in which the red plane marks τ = 0.3; pixels whose intensity exceeds this plane are counted toward DSI. Below, the 2D thresholded map shows counted pixels ( I norm > τ) in condition color and uncounted pixels ( I norm ≤ τ) in dark gray, with the DSI fraction bar indicating the proportion of counted pixels. DSI increases from the compact (DSI = 0.367) to the decompact state (DSI = 0.792).

Article Snippet: For live-cell imaging, dHL-60 cells (day 6 or 7 post-differentiation) were stained with 1μM SPY650-DNA (Cytoskeleton, CY-SC501; reconstituted in DMSO) for 1 hour at 37°C in a humidified 5% CO 2 incubator.

Techniques: Fluorescence, Staining

Journal: bioRxiv

Article Title: Benchmarking three simple DNA staining-based image metrics for live-cell tracking of chromatin organization

doi: 10.64898/2026.03.30.715467

Figure Lengend Snippet: (A) Representative time-lapse fluorescence images of dHL-60 cell nuclei stained with SPY650-DNA during NETosis stimulation (4 µM ionomycin). Top row (blue box): non-NETing cell at 0, 120, and 240 min from movie start. Bottom row (red box): NETing cell at −8, 0, and 82 min relative to nuclear rounding onset. NETosis stages are indicated below each image. Scale bars, 5 µm. (B–D) Population-level time series of CV (B) , 1-Gini (C) , and DSI (D) for NETing (red, n = 42) and non-NETing (blue, n = 28) cells. Solid lines indicate population means; shaded regions indicate ±1 SD. Cell trajectories were aligned to normalized time (0 = nuclear rounding onset, 1 = frame immediately preceding nuclear envelope rupture for NETing cells; 0 = movie start, 1 = movie end for non-NETing cells). Gray dashed lines show p-values (right y-axis) at each normalized time point (BH correction); the gray solid line indicates p = 0.05. DSI showed statistically significant separation between NETing and non-NETing trajectories at 62.2% of normalized time points, compared with 17.4% for 1-Gini and 2.9% for CV. (E–G) Per-cell trajectory-averaged CV (E) , 1-Gini (F) , and DSI (G) for non-NETing and NETing populations. Each dot represents one cell’s mean metric value across its entire trajectory. Horizontal bars indicate population mean; error bars indicate ±1 SD. Only DSI showed a statistically significant difference between the two populations (p = 3.64 × 10 -2 , *), whereas CV (p = 6.19 × 10 -1 , n.s.) and 1-Gini (p = 9.00 × 10 -1 , n.s.) did not. Mean ± SD values are shown below each panel.

Article Snippet: For live-cell imaging, dHL-60 cells (day 6 or 7 post-differentiation) were stained with 1μM SPY650-DNA (Cytoskeleton, CY-SC501; reconstituted in DMSO) for 1 hour at 37°C in a humidified 5% CO 2 incubator.

Techniques: Fluorescence, Staining

Journal: bioRxiv

Article Title: Benchmarking three simple DNA staining-based image metrics for live-cell tracking of chromatin organization

doi: 10.64898/2026.03.30.715467

Figure Lengend Snippet: (A) Representative fluorescence images of DNA (DAPI, displayed in grayscale; top) and Tn5 transposase signal (Tn5-Oligo-Alexa Fluor 488, displayed in red; bottom) in fixed dHL-60 cells at indicated NETosis stages. NETosis stage was classified based on nuclear morphology and Lamin B Receptor staining (not shown). Scale bar, 5 µm. (B–D) Scatter plots of CV (B) , 1-Gini (C) , and DSI (D) versus Tn5 integrated intensity for individual fixed cells at two NETosis stages: before nuclear rounding (light, n = 297) and nuclear rounding (dark, n = 95). Spearman’s ρ and associated p-values are shown above each panel. Insets zoom into the 5th–95th percentile range of each metric on the x-axis (dashed lines indicate the zoomed region) with a linear regression line. All three metrics showed statistically significant monotonic correlations with Tn5-based accessibility. CV exhibited the strongest association (ρ = −0.504), followed by DSI (ρ = 0.382) and 1-Gini (ρ = 0.258); the negative sign of CV reflects its inverse relationship to chromatin decompaction.

Article Snippet: For live-cell imaging, dHL-60 cells (day 6 or 7 post-differentiation) were stained with 1μM SPY650-DNA (Cytoskeleton, CY-SC501; reconstituted in DMSO) for 1 hour at 37°C in a humidified 5% CO 2 incubator.

Techniques: Fluorescence, Staining