transwell insert  (Servicebio Inc)

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

Journal: Bioactive Materials

Article Title: Reconstructing the ischemic osteogenic microenvironment through hierarchical scaffolds orchestrating Mg 2+ signaling and neuropilin-1–mediated angiogenesis

doi: 10.1016/j.bioactmat.2026.02.031

Figure Lengend Snippet: Evaluation of angiogenic potential induced by Mg 2+ and NRP-1. ( A ) Scratch assay in three different microenvironments. ( B ) Transwell assay in three different microenvironments. ( C ) Quantitative analysis of wound healing area. ( D ) Quantitative analysis of number of migration cells. ( E ) Immunofluorescence staining of VEGFA and FGF2, with DAPI for nuclear staining and F-actin for cytoskeleton labeling. ( F ) Tube formation evaluation in four different microenvironments. ( G ) Quantitative analysis of number of junction. ( H ) Quantitative analysis of Flu intensity. ( I ) Western-blot analysis of VEGFA, FGF2, and Nr4a1. ( J-L ) Quantitative analysis of relative protein expression of VEGFA, FGF2, and Nr4a1. Data are presented as mean values ± s.d. (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (one-way ANOVA).

Article Snippet: For the Transwell assay, BMSCs were seeded in the upper chambers of Transwell inserts (8.0 μm pore size; Servicebio, China), while different culture conditions were applied in the lower chambers according to the experimental groups.

Techniques: Wound Healing Assay, Transwell Assay, Migration, Immunofluorescence, Staining, Labeling, Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: Multi-omics profiling identifies ADAM9 as a key efferocytosis driver in lung adenocarcinoma

doi: 10.3389/fimmu.2026.1772167

Figure Lengend Snippet: Effects of ADAM9 knockdown in macrophage cells. (A) Relative mRNA expression of ADAM9 in LUAD tissues and adjacent tissues. (B) ADAM9 expression in LUAD tissues evaluated by IHC. (C, D) ADAM9 expression in M2 macrophages transfected with siRNA. (E) RT-qPCR analysis of M2 polarization markers in si-ADAM9-transfected M2 macrophages. (F) ATP content assay in M2 macrophages transfected with si-ADAM9. (G) ADAM9 mRNA expression in normal bronchial epithelial cells and LUAD cell lines. (H) Transwell migration assay showing the migratory ability of A549 and H1975 cells co-cultured with M2 macrophages. (I, J) EdU assay detecting the proliferative capacity of LUAD cells co-cultured with M2 macrophages. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were pre-cultured in serum-free medium for 48 h. Then, 2.0×10 4 cells were seeded in the upper chamber of a transwell insert (NEST Biotechnology, Wuxi, China), while the lower chamber was filled with RPMI-1640 medium containing 10% FBS (Bioland, China).

Techniques: Knockdown, Expressing, Transfection, Quantitative RT-PCR, Transwell Migration Assay, Cell Culture, EdU Assay