Fig. 6: HYDRA-like hydrogels on an imaging-grade 384-well plate.

a Hydrogels were cast in a 384-well plate by using 10% w/v FG/0.5% w/v TG. Hydrogel precursor solutions were mixed with fluorescent beads (shown in magenta) to visualize the gel volume. An automatic quality control analysis was performed to evaluate hydrogel morphology and check if the sample touched the well walls. 91% of the samples resulted in well-deposited planar hydrogels (green boxes), 6% of them formed a meniscus due to touching the walls (red boxes), while 3% of samples appeared with a concave shape (yellow boxes). “x” symbols on well shows wrong classification done by the automatic analysis. Well width: 3.7 mm. b 3D rendering of HaCaT cells (actin in gray) on the hydrogel surface (fluorescent beads in magenta). (c) 18-hour live fluorescence imaging experiment of HaCaT cells on hydrogels. HaCaT cells express fluorescent sensors for actin (in gray) and the cell cycle (cyan nuclei in G1 phase in cyan, nuclei in S/G2/M phase in magenta). Segmentation masks for selected cells and tracks backward in time are shown. Mask outlines were colored manually on TrackMate according to the cell cycle phase. A tracking diagram is shown where each spot represents a cell at a specific phase of the cell cycle at each time point. The dashed white box indicates the tracking lineage of the highlighted cells. d Max intensity projections of the XY, XZ, and YZ planes from a z-stack showing a cluster of cells on hydrogels (actin in gray, tubulin in green, nuclei in cyan and magenta). (i) The red square box zooms in on three cells undergoing mitosis, evidenced by mitotic spindles in green. (ii) The white dashed box shows the actin and tubulin structures of cells well spread on the hydrogel surface. Zooms were adjusted by applying a Sharpening filter in FIJI³⁰. Scale bars: 25 µm.