Fig. 2: Integration of fluorescent-based nuclear imaging with the Seahorse metabolic flux assay. | Communications Biology

Fig. 2: Integration of fluorescent-based nuclear imaging with the Seahorse metabolic flux assay.

From: High-content fluorescence imaging with the metabolic flux assay reveals insights into mitochondrial properties and functions

Fig. 2

a T3M4 cells were seeded in increasing densities in the Seahorse XF plate and processed through the XF assay for OCR and plotted. n = 8–10 biologically independent samples. b OCR values were corrected for and plotted against manually counted cell seeding densities. Statistical significance determined by one-way ANOVA; *p = 0.038, **p = 0.001. n = 7–10 biologically independent samples. c OCR measurements were corrected for fluorescently counted nuclei (i.e., Hoechst+ object) and plotted against seeded cell densities. n = 8 to 10 biologically independent samples. d OCR data (empty circle) extrapolated from individual columns of the XF plate display significant variation at the edge columns (i.e., columns 2 and 11), while no changes are observed in nuclei distribution (gray circle). Statistical significance determined by two-way ANOVA; **p = 0.008, ***p = 0.000013, ***p = 5 × 10−7. n = 8 biologically independent samples. e Cell cycle analysis distribution of taxol treated cells, identified through nuclei fluorescent imaging (e.g., Hoechst+ object). Statistical significance determined by one-way ANOVA, ***p < 0.001 (complete p-values can be seen in Supplementary Table 3). Data presented as the average + /− s.d. of n = 10 biologically independent samples. f Post hoc distribution analysis of cells treated with increasing concentrations of taxol and their respective nuclei staining intensity (A.U. arbitrary fluorescent units) n = 10 biologically independent samples. g Image analysis of nuclei size via fluorescently labeled nuclei post taxol treatment; n = 20 biologically independent samples. h OCR values corrected for fluorescently identified nuclei are increased post taxol treatment; n = 14 to 16 biologically independent samples. Statistical significance in g, h determined by Student’s t-test; g ***p = 10−14; h ***p = 4 × 10−9. All experiments were the result of ≥2 independent experiments. All measurements were obtained from distinct samples.

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