Phenotypic characterization of multicellular 3D spheroid’s morphology, viability, and cell proliferation. (A) Microscopic images of multicellular 3D spheroids composed of BEAS-2B, MRC-5, and THP-1 (BMT spheroid); A549, MRC-5, and THP-1 (AMT spheroid); and NCI-H460, MRC-5, and THP-1 (HMT spheroid) on day 4, day 7, and day 10. (B) Measurement of spheroid’s diameter on day 4, day 7, and day 10. (C) Schematic diagram (upper panel) and microscopic images (lower panel) of spheroid development at different time points displaying initial loose association of cells (days 0-3) followed by tight aggregates (days 4-8) leading to compact spheroid formation (day 9onwards). (D, E) RT-qPCR analysis of E-cadherin(D) and fibronectin(E) mRNA levels in BMT, AMT, and HMT spheroids on day 0, day 7, and day 10. (F, G)Calcein-AM and propidium iodide (PI) staining (F), and the quantification of Calcein-AM-stained live cells (G) in BMT, AMT, and HMT spheroids on day 4, day 7, and day 10(n=3). (H) Determination of acid phosphatase(APH) activity in developed spheroids(n=3). (I) RT-qPCR analysis of cell proliferation marker MKi67 in BMT, AMT, and HMT spheroids on day 4, day 7, and day 10(n=3). (J) Immunofluorescence images of Ki67 protein in day 10 spheroids section. DAPI was used for nuclei counterstaining. GAPDH is a loading control for RT-qPCR studies. Fold change for E-cadherin, fibronectin, and MKi-67gene expression was calculated keeping the spheroid cell cocktail (BEAS-2B/MRC-5/THP-1, A549/MRC-5/THP-1, and NCI-H460/MRC-5/THP-1) at day 0 as control. All experiments were performed in triplicate (n=3). Data represented as mean ± S.D. 2D vs BMT over time *p< 0.05, **p< 0.01, ***p< 0.001; BMT vs AMT over time #p< 0.05, ##p< 0.01, ###p< 0.001; BMT vs HMT over time §p< 0.05, §§p< 0.01, §§§ p< 0.001; ns, non-significant.
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