Journal: STAR Protocols

Article Title: Protocol for Seahorse 3D Mito Stress assay in patient-derived atypical teratoid rhabdoid tumor CHLA-05-ATRT single neurospheres

doi: 10.1016/j.xpro.2026.104515

Figure Lengend Snippet: Oxygen consumption rate (OCR) in the presence or absence of spheroids (A) Incucyte image of spheroid in the center of the microplate well at the end of the assay and (B) the corresponding OCR kinetic graph showing a response after drug injections as expected. (C) Image of a microplate well with no spheroid detected at the end of the assay and (D) corresponding OCR kinetic graph showing no response after drug injections.

Article Snippet: Incucyte Organoid Analysis Software Module , Sartorius , 9600–0034.

Techniques:

Journal: STAR Protocols

Article Title: Protocol for Seahorse 3D Mito Stress assay in patient-derived atypical teratoid rhabdoid tumor CHLA-05-ATRT single neurospheres

doi: 10.1016/j.xpro.2026.104515

Figure Lengend Snippet: Effect of coating timing on spheroid retention in microplate wells (A) Incucyte image of a poly-lysine coated microplate with spheroids transferred on the same day as the assay, taken before the assay. (B) Image of the same plate taken after the assay, showing that the spheroids were displaced and/or destroyed during the assay. (C) Image of a poly-lysine coated microplate with spheroids transferred the day before the assay, taken before the assay and (D) after the assay, showing that the spheroids were intact.

Article Snippet: Incucyte Organoid Analysis Software Module , Sartorius , 9600–0034.

Techniques:

Journal: Communications Biology

Article Title: An ontogeny-cytokine code determines macrophage response polarity and tumor outcomes

doi: 10.1038/s42003-026-09853-y

Figure Lengend Snippet: A Experimental workflow used to generate reference macrophage states. BM precursors were differentiated with GM-CSF or M-CSF and skewed with IFN-γ, IL-4, IL-10, or TGF-β, yielding eight cytokine × ontogeny macrophage states. Macrophages were harvested on day 7 for live-cell imaging, morphological analysis, and T-cell co-culture assays. Color code: Control (gray), IFN-γ (blue), IL-4 (green), IL-10 (yellow), TGF-β (orange-red). B Time-lapse live microscopy of Arg1-YFP (green) and Spp1‑IRES‑tdTomato (red) reporter macrophages. Curves plot mean fluorescence over 100 h for each cytokine in M-CSF and GM-CSF cultures (representative of three independent experiments; six imaging fields per well per condition were analyzed). Timepoint 0 represents the time of cytokine stimulation (day 3 of culture). IL-4 induces a progressive ARG1 signal only in M-CSF macrophages; IL-10 (and weakly TGF-β) induces SPP1 almost exclusively in GM-CSF macrophages; unpolarized and IFN-γ macrophages maintain low expression of both reporters throughout. C Representative fluorescence micrographs showing label-free cell classification based on automated quantification of cell area (primary metric for GM-CSF macrophages) and eccentricity (primary metric for M-CSF macrophages). Images were acquired using live-cell imaging (Incucyte) and analyzed using the Advanced Label-Free Classification Module. Pseudo-color overlay indicates morphometric values: cyan = low (round cells with low area/eccentricity), magenta = high (elongated or large cells with high area/eccentricity). Representative images shown for each cytokine treatment in both M-CSF and GM-CSF cultures. Scale bar = 200 µm. D Z -scored classification data from four independent biological replicates (open circles). Eccentricity (primary morphological discriminant for M-CSF macrophages) and cell area (primary morphological discriminant for GM-CSF macrophages). Z -scores were calculated across all conditions to enable direct comparison. E Flow-cytometric contour plots for CD11b and MHC-II expression in GM-CSF macrophages across all treatment conditions and CFSE dilution histograms of OT-II specific CD4+ T cells co-cultured with peptide-pulsed GM-CSF macrophages. F Bar graphs of the cumulative data showing the fraction of OT-II CD4+ T cells that underwent proliferation. Data represent mean ± SEM from n = 6 independent cultures, one-way ANOVA Tukey‒Kramer posttest corrected for multiple comparisons (Control vs IL‑10 p < 0.0001; control vs TGF‑β p < 0.0001; IFN‑γ vs IL‑10 p < 0.0001; IFN‑γ vs TGF‑β p < 0.0001; IL‑4 vs IL‑10 p < 0.0001; IL‑4 vs TGF‑β p < 0.0001. All other comparisons, including control vs IFN‑γ, control vs IL‑4, IFN‑γ vs IL‑4, and IL‑10 vs TGF‑β were not significant ( p > 0.05; ns).

Article Snippet: The area and fluorescence intensities of the images were measured using the IncuCyte Spheroid Software Module (Sartorius).

Techniques: Live Cell Imaging, Co-Culture Assay, Control, Microscopy, Fluorescence, Imaging, Expressing, Comparison, Cell Culture

Journal: Communications Biology

Article Title: An ontogeny-cytokine code determines macrophage response polarity and tumor outcomes

doi: 10.1038/s42003-026-09853-y

Figure Lengend Snippet: A GFP‑MC38 cells were cultured alone or mixed 1:1 with cytokine‑polarized M‑CSF or GM‑CSF macrophages in ultra‑low‑attachment plates and imaged for 10 days. Macrophages were pre-polarized for 7 days as described in Fig. , then washed and mixed with tumor cells. Spheroids formed within 24 h and were imaged every 2–4 h for 10 days using an Incucyte S3 live-cell analysis system. GFP fluorescence intensity integrated across the entire spheroid area was used as a real-time readout of spheroid size. For each treatment condition 9–10 spheroids were analyzed. B M-CSF macrophages : Integrated GFP fluorescence intensities across the spheroid area over time (mean ± SE of 8–10 spheroids analyzed within one representative experiment) and area under the curve (AUC), reflecting cumulative spheroid growth over the 10-day period. Individual data points (open circles) show replicate values. one-way ANOVA Dunnett posttest corrected for multiple comparisons (Control vs IFN‑γ p = 0.0110, control vs all other cytokines p < 0.0001. C Representative fluorescence micrographs for each condition from the time course in B . Each image shows a single spheroid. Scale bar = 0.5 mm. D GM-CSF macrophages: Time-course analysis and AUC quantification identical to B . Data are the mean ± SE of 9 biologically independent spheroids from one independent experiment. Individual data points represent individual spheroids (Control vs IFN-γ p = 0.9993 (ns), control vs IL-4 p = 0.0269, control vs all other cytokines p < 0.0001). E GM-CSF macrophages: Representative fluorescence micrographs for each condition from the time-course experiment in D . Each row shows a representative spheroid over time. Scale bar = 0.5 mm.

Article Snippet: The area and fluorescence intensities of the images were measured using the IncuCyte Spheroid Software Module (Sartorius).

Techniques: Cell Culture, Cell Analysis, Fluorescence, Control

Journal: Communications Biology

Article Title: An ontogeny-cytokine code determines macrophage response polarity and tumor outcomes

doi: 10.1038/s42003-026-09853-y

Figure Lengend Snippet: A GFP‑MC38 cells were cultured alone or mixed 1:1 with cytokine‑polarized M‑CSF or GM‑CSF macrophages in 96-well ultra‑low‑attachment or 24-well microwell plates. Three days post formation, spheroids were embedded in a laminin-rich matrix ( t = 0 h) and invasion was visualized by Incucyte live-cell imaging or confocal microscopy. B Confocal micrographs acquired 24 h after embedding show dense, finger‑like projections invading into the matrix in spheroids containing protumoral macrophages (IL-4 polarized M-CSF macrophages (green), IL-10 polarized GM-CSF macrophages (yellow)). Scale bar = 100 µm. C M-CSF macrophages: Quantitative spheroid invasion assay. Cell invasion was measured with an Incucyte live cell imaging system every 4 h. The invading cell front was automatically segmented and quantified over time. Data are the mean ± SE of 3 biologically independent spheroids analyzed within one representative experiment. AUC quantification was analyzed by one-way ANOVA Dunnett posttest corrected for multiple comparisons (Control vs IFN-γ p = 0.6900 (ns), control vs IL-4 p < 0.0001, control vs IL-10 p = 0.3239 (ns), control vs TGF-β p < 0.0001, control vs TC p = 0.6665 (ns)). Representative inverted bright field images for each condition are shown. Scale bar = 0.5 mm. D GM-CSF macrophages: Analysis identical to C ( n = 4 replicates per condition) (Control vs IFN-γ p = 0.0714 (ns), control vs IL-4 p = 0.7959 (ns), control vs IL-10 p < 0.0001, control vs TGF-β p < 0.0001, control vs TC p = 0.0954 (ns)). Scale bar = 0.5 mm.

Article Snippet: The area and fluorescence intensities of the images were measured using the IncuCyte Spheroid Software Module (Sartorius).

Techniques: Cell Culture, Live Cell Imaging, Confocal Microscopy, Invasion Assay, Control