Bz7000 microscope

To evaluate the time requirements for scan imaging, 1 × 10⁴ cells were seeded. After 11 h, a total of 265 images were captured every 30 mins for 6 days. The data were plotted in graph format at 12 h intervals.
To understand the variation in cell density based on the observation position, a simulation was conducted. Sampling was performed from 25 fields of view, each of the same size as a typical microscopic image (using a field of view with a 4× lens on the Keyence BZ7000 microscope). The average cell count for each image was calculated to estimate the cell count for the entire dish. The discrepancy between the estimated and the actual cell counts was then determined. The data from a single field represented the time series changes in the estimated number of cells at each fixed point within the 25 fields of view. Data from 3 to 20 fields of view were processed by considering five combinations of sampling fields.

Spheroids were fixed with cold 4% PFA in PBS. After blocking with Block Ace (DS Pharma Biomedical), the spheroids were permeabilized with 0.1% saponin and stained with FITC-conjugated anti-CD44 mAb (clone; IM7, BioLegend) plus biotin-conjugated hyaluronan-binding protein (HABP) (Seikagaku Corp.) After a PBS wash, the spheroids were stained with Alexa594-conjugated streptavidin and 4′, 6-diamidino-2-phenylindole (DAPI). For the analyses of Ki-67 expression, anti-Ki-67 mAb (Becton Dickinson) and Alexa594-conjugated anti-mouse IgG (Molecular Probes) were used. In some experiments, spheroids were treated with hyaluronidase (100 U/ml; Sigma) and incubated with FITC-labeled HA (FL-HA; Seikagaku Corp.) in the presence or absence of anti-CD44 mAb (clone; BRIC 235). Fluorescent images were taken with the BZ-7000 microscope (Keyence). The specificity of the HABP was confirmed by hyaluronidase treatment before HABP staining.

The cells were washed with phosphate-buffered saline (PBS), resuspended (1.0 × 10⁴ cells/ml) in DMEM/F-12 (Gibco) supplemented with 50 ng/ml EGF (R&D), 10 ng/ml bFGF (R&D), 25 μg/ml insulin (Gibco), and 2% B27 supplement (Gibco), and cultured for 4 days in ultra-low attachment surface plates (Corning) [21 (link)]. Images of spheroids were acquired with a BZ-700×0 microscope (Keyence), and the Heywood diameter of the spheroids was determined. The number of spheroids (>30 or >100 μm in Heywood diameter) in 5 low-power fields (×100) was counted [35 (link), 37 (link)]. Frequency of spheroids (>100 μm) (%) was calculated as follows: [the number of spheroid (>100 μm)/the total number of spheroids (>30 μm)] × 100. In some experiments, 4-methylumbelliferone (4MU) (100 μΜ) (Sigma), hyaluronan (100 μg/ml) (Seikagaku Corp.), SB431542 (10 μm) (R&D), anti-CD44 monoclonal antibody (mAb) (clone: BRIC235, 10 μg/ml) (IBGRL), or anti-activin A (betaA subunit) mAb (Clone: #69403, 10 μg/ml) (R&D) was added to the culture medium.