Fig 1: Population-level comparison of 3D vs. 2D cultures. a Representative images of spheroids in the anchors and 2D cells stained for LIVE/DEAD, ALB and BrdU. b Actin organization in one spheroid on chip (confocal slices at two vertical positions), and cells in 2D. c Evolution of the viability of the spheroids on chip and of the cells in 2D, during a 7-day culture period (n chips = 3; n 2D = 4). d Quantification of the percentage of BrdU+ cells in spheroids on chip and cells in 2D, over a 7-day culture period (n chips = 5; n 2D = 4). e ALB productivity at day 4 by spheroids on chip and cells in 2D, measured by ELISA on the supernatant (n chips = 2; n 2D = 3). f RT-qPCR analysis of relative ALB expression to GADPH (?Ct), in 3D and in 2D (n chips = 3; n 2D = 3) g RT-qPCR analysis of ALB expression (Relative RNA expression), in 3D and in 2D (n chips = 3; n 2D = 3). h Representative gel of RT-PCR analysis of ALB and GAPDH expression, in 3D and in 2D. All scale bars are 50 µm. Error bars show the standard deviation of each population. *p < 0.05, unpaired two-sample two-tailed Student’s t-tests
Fig 2: Multiscale cytometry on chip. a Measured amplitude of intracellular ALB. Each dot represents one spheroid and each color represents one chip. The mean value for each chip is shown by a larger dot with the same color. Solid black line: average ALB signal at each time point. D + 1/D + 3/D + 5/D + 7: n = 5/4/6/4 chips; N = 4,925 spheroids. b, c Normalized ALB signal for each spheroid vs. b the spheroid diameter and c the shape index. The normalization was performed by dividing a specific spheroid ALB intensity value by the average ALB signal within a chip. By this way, it was possible to compare ALB production of spheroids from different chips. The histograms show the distribution of the data along the different axes. Blue curves show Gaussian fits, dotted blue line: mean, solid blue line: median. N spheroids = 4,925, N cell aggregates = 506, and N units = 389. d Representative images for a spheroid in bright field (top), stained for DAPI (middle), and for ALB (bottom). R represents the effective spheroid radius and r the radial coordinates of each detected peak in the fluorescent signal within the spheroid. Scale bar is 50 µm. e Mean distance between each cell and its 3 nearest neighbors (n cells = 128,973, N spheroids = 6,236). dotted magenta line: first decile and dotted cyan line: last decile of the distances. f Corresponding histograms of all the nuclei and of the two gated populations of part e. (magenta=below the first decile; cyan=above the last decile). Dashed lines give the median values. g Confocal image of the mid-plane of a spheroid stained for phalloidin (red) and DAPI (blue). Dashed lines emphasize two representative cells. h Normalized ALB vs. distance from spheroid center (n ALB maxima = 87,609, N spheroids = 4,654). i Corresponding histogram for gated populations. j Confocal image of the mid-plane of a spheroid stained for ALB (green). White dashed line shows the spheroid edge. Non significant N.S., ***p < 0.001. a, b Kruskal–Wallis ANOVA followed by Mann–Whitney U-tests with Sidak’s correction for multiple comparisons. c Welch’s ANOVA followed by Games-Howell post hoc procedure
Supplier Page from Abcam for Rat Albumin ELISA Kit