Journal: Bio-protocol

Article Title: 3D Iterative Immunofluorescence Imaging on Whole-Mount Samples

doi: 10.21769/BioProtoc.5838

Figure Lengend Snippet: (A) Sample and plate preparation steps for mounting before performing the 3D-4i multiplexing protocol. (B) Visualization of the zebrafish embryos mounted inside the imaging plate well. Example fluorescence image in side view (x–z) acquired with a spinning disk confocal microscope and 1 μm z-step size (arrow depicts imaging direction from objective). Scale bar: 50 μm. (C) Example overview of a 96-well imaging plate with 5–6 zebrafish embryos mounted each in 24 individual wells. DAPI signal with color corresponding to cycle 1 (red) and 4 (blue) of the 3D-4i multiplexing protocol. Total number of embryos is 138; the number of detached embryos during a four-cycle 3D-4i protocol is 1 (second column from the left, third row from the top).

Article Snippet: Laboratory centrifuge with rotors compatible with 96-well plate inserts (e.g., Eppendorf, model: 5810R) 8.

Techniques: Multiplexing, Imaging, Fluorescence, Spinning Disk, Confocal, Microscopy

Journal: Nature Communications

Article Title: Cell-derived lactate triggers local hydrogelation and phenotype remodelling

doi: 10.1038/s41467-026-77702-x

Figure Lengend Snippet: Unless stated otherwise, data are mean ± s.d. Statistics: unpaired two-tailed Welch’s t -test; P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P > 0.05 (ns, not significant). a Transwell assay schematic: A549 cells in the lower well; AlgTA + lactate oxidase (LOx) + horseradish peroxidase (HRP) in the insert. Cell-secreted lactate diffuses into the insert and is oxidised by LOx to generate H 2 O 2 , which HRP uses to oxidatively couple AlgTA phenols and form a gel (see also Fig. ). Created in BioRender. Braun, M. (2026) https://BioRender.com/569s2eu . b Representative fluorescence micrographs of A549 cells at days 1–3. Stains: Calcein AM (live, green), NucBlue (nuclei, blue), propidium iodide (dead, red), n = 3 independent experiments. Scale bars, 100 µm. c Resazurin-based metabolic activity ( n = 9 wells) from 3 independent experiments. Shown P -values: day 1, P = 0.054, day 2, P < 0.001; day 3, P < 0.001. d Relative proteome profiling of selected tumour-associated proteins in AlgTA/LOx/HRP-treated cultures at day 2 versus matched controls ( n = 3 biological replicates, 10 wells each, 2 on-membrane replicates). Shades of red indicate decreased, and shades of green indicate increased relative protein abundance. e Evolution of mechanics and transport: Young’s modulus E measured by nanoindentation ( n = 9 indentations from 3 gels; see the “Methods” section) and fluorescence correlation spectroscopy (FCS) diffusion-time ratio ( τ gel / τ buffer ) for gels formed at days 1–3 after equilibration in tracer-containing PBS ( n = 4 positions per gel). f Lactate concentration under the indicated conditions at days 1–3. ( n = 6 samples from 2 independent experiments). Shown P -values: day 1, P = 0.005; day 2, P < 0.001; day 3, P < 0.001. g Extracellular H 2 O 2 quantified by a fluorimetric hydrogen peroxide assay at days 1–3 ( n = 4 samples).

Article Snippet: 25.000 A549 cells were seeded in a 24-well plate and cultured using 600 μL Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose, Gibco 31053-028) supplemented with 10% dialysed foetal bovine serum (FBS, Gibco A33820-01) and 1% Penicillin/Streptomycin for 24 h. The medium was exchanged, the gel precursor-filled Transwell (Greiner Bio-One 662641) was added, and the experiment started.

Techniques: Two Tailed Test, Transwell Assay, Fluorescence, Activity Assay, Profiling, Membrane, Quantitative Proteomics, Spectroscopy, Diffusion-based Assay, Concentration Assay, H2O2 Assay

Journal: Nature Communications

Article Title: Cell-derived lactate triggers local hydrogelation and phenotype remodelling

doi: 10.1038/s41467-026-77702-x

Figure Lengend Snippet: Unless stated otherwise, data are mean ± s.d. (day 2). Statistics: unpaired two-tailed Welch’s t -test; P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P > 0.05 (ns, not significant). Formulation: 3 wt% AlgTA, 10 U mL −1 HRP, 1 U mL −1 LOx; control: DMEM supplemented with FBS. a Communication cascade: spheroid-secreted lactate is converted by LOx/HRP to H 2 O 2 , driving phenol coupling and in situ AlgTA gelation around the spheroid (schematic) and photograph of encapsulated spheroids. Scale bar, 2 mm. Created in BioRender. Braun, M. (2026) https://BioRender.com/569s2eu . b Representative fluorescence micrographs of A549 spheroids: F-actin (phalloidin, green) and nuclei (NucBlue, blue), n = 3 independent experiments. Top: control (regular medium). Bottom: AlgTA/LOx/HRP, showing actin-rich protrusions. Scale bars, 100 µm. c Calibration of hydrogel crosslinking with defined lactate concentrations (green). Correlation between the diffusion-time ratio of Alexa488-BSA ( τ gel / τ buffer ) and known concentrations of lactate (0–10 mM sodium lactate). d Spatial transport mapping by FCS: diffusion-time ratio ( τ gel / τ buffer ) and apparent lactate-equivalent concentration ( c lac , eq ) versus distance from the spheroid surface. Individual values were taken at three parallel offset distances and are shown with a line representing the mean. e Proteomics (3D, day 2): relative abundance of selected tumour-associated proteins in AlgTA/LOx/HRP versus control ( n = 3 biological replicates, 12 spheroids each, 2 on-membrane replicates). Red indicates decreased, and green indicates increased relative protein abundance. f Proteomic profiling of treated AlgTA/LOx/HRP 2D Transwell and 3D spheroid cultures. Filled bars indicate the condition showing the stronger relative response. 3D: 3 biological replicates, 12 spheroids each, 2 on-membrane replicates; 2D: n = 3 biological replicates, 10 wells each, 2 on-membrane replicates. g Representative spheroid diameter with and without AlgTA/LOx/HRP; 16 spheroids per condition. Shown P -values: AlgTA/LOx/HRP day 0 vs day 2, P = 0.546; control day 0 vs day 2, P < 0.001; AlgTA/LOx/HRP day 2 vs control day 0, P < 0.001, n = 3 experiments. h Metabolism under encapsulation ( n = 3 independent experiments): CellTiter-Glo® 3D (ATP, n = 9 wells, P < 0.001) and resazurin (NADH, n = 9 wells, P < 0.001) assays.

Article Snippet: 25.000 A549 cells were seeded in a 24-well plate and cultured using 600 μL Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose, Gibco 31053-028) supplemented with 10% dialysed foetal bovine serum (FBS, Gibco A33820-01) and 1% Penicillin/Streptomycin for 24 h. The medium was exchanged, the gel precursor-filled Transwell (Greiner Bio-One 662641) was added, and the experiment started.

Techniques: Two Tailed Test, Formulation, Control, In Situ, Spheroids, Fluorescence, Diffusion-based Assay, Concentration Assay, Membrane, Quantitative Proteomics, Profiling, Encapsulation