Multivariable logistic regression Models 1 and 2.

<p>ROC: receiver operating characteristic. AUC: area under the curve. Walking conditions: UP = Usual pace, FP = Fast pace, SP = Slow pace, CW = count walk, AW = animal walk.</p

Multivariable logistic regression Models 1 and 2.

<p>ROC: receiver operating characteristic. AUC: area under the curve. Walking conditions: UP = Usual pace, FP = Fast pace, SP = Slow pace, CW = count walk, AW = animal walk.</p

Dotplot: Multivariate regression model for association between dementia stages (CDR code) and gait variables adjusted for gender in five walking conditions for all participants and for age-stratified groups (two-way ANOVA).

<p>Dotplot_all: Main effect test of CDR on gait parameter, for all ages combined. A 2-way ANOVA model was fitted, with main effects for CDR score and gender but without the interaction term. The dotplot shows the negative logarithm (10-based) of the p-value for the main effect of CDR score.Dotplot_5070: Main effect test of CDR score on gait parameter (age 50 to 70). Dotplot_7080: Main effect test of CDR score on gait parameter (age 70–80). Dotplot_80plus: Main effect test of CDR score on gait parameter (above age 80).</p

Dotplot: Association between dementia stages (CDR code) and gait variables (one-way ANOVA).

<p>Dotplot_all: Association between CDR score and gait (all ages combined). The dotplot shows the negative logarithm (10-based) of the p-values for the one-way ANOVA between gait parameter and CDR score. Strong associations with a small p-value correspond to large values of the–log(p). Each line in the plot corresponds to one gait parameter. On each line, five dots are shown for the 5 walking conditions. Dotplot_5070: Association between CDR score and gait (age 50 to 70). Dotplot_7080: Association between CDR score and gait (age 70–80). Dotplot_80plus: Association between CDR score and gait (above age 80). CDR: Clinical Dementia rating. DTC: dual task cost.</p

Time-dependent migratory pattern of MDA-MB-231 and A549.

<p>A. MDA-MB-231 (<i>left</i>) and A549 (<i>right</i>) cell migration profiles, detected by Transwell experiments (<i>black</i>) and <i>xCELLigence</i> (<i>red</i>). Graphs represent scaled signals (0–1) of net chemoattraction after subtraction of the random migration signal (<i>empty squares</i> in panel A, B), with associated Spearman's Rho values. All results originate from three independent duplicate experiments ± SD. B. Normalization procedure of migration patterns. Raw data (<i>left panel</i>) were normalized to a (0–1) scale (<i>middle panel</i>) through division of all data by the maximum value obtained in three independent experiments. Subsequently, random migration (SF) signals (<i>triangle markers</i>) were subtracted from the positive (FBS) control counterparts (<i>circle markers</i>) per experiment to obtain a pure chemotactic signal (<i>right panel</i>). Example shown is the migratory pattern of MDA-MB-231 cells estimated by pixel area calculation in three experiments (exp 1 - <i>red</i>, exp 2 - <i>green</i>, exp 3 - <i>black</i>). Triangle and circle markers represent negative (SF) and positive (FBS) control data respectively. C. ImageJ-based picture processing. Original pictures were color thresholded to obtain a binary image displaying cellular content as saturated black areas on a white background. Thresholded images were masked to exclude non-cellular particles from the final area calculation. Pictures shown are migrated MDA-MB-231 cells after four hours (<i>top row</i>) and 16 hours (<i>bottom row</i>) of incubation. D. Migratory behavior of MDA-MB-231 cells toward medium+FBS (positive control – <i>filled squares</i>) and background migration (<i>empty squares</i>) as detected by conventional Transwell experiments at ten time points spread over 24 hours of incubation. Area calculation (<i>left</i>) of stained cells and optical density (OD – <i>middle</i>) were compared to the <i>xCELLigence</i> migration pattern, reconstructed from the original high-resolution plot by extrapolating data from the corresponding time points (<i>right</i>). All results represent original data from three independent duplicate experiments ± SD. Picture string (obj. 2.5×) shows migratory status of MDA-MB-231 cells, stained as described, at five different stages during 24 hours of incubation. E. Same as (D) for A549 cells.</p

Prestatistical data processing.

<p>A. Schematic depiction of processing kinetic data generated by SRB, <i>xCELLigence</i> and Transwells. Raw data with high time resolution (<i>filled</i> and <i>empty circles</i>), resulting from independent <i>xCELLigence</i> experiments (1, 2, 3 and <i>grey arrows</i>) are reduced to a lower time resolution by selecting only the data points corresponding with the time points of endpoint detection (<i>filled circles</i> only). Subsequently, data have been normalized by dividing all values by the highest value recorded over all experiments per method, resulting in a modified Y-axis scale that ranges from 0 to 1. Finally, the normalized data have been averaged with calculation of SD for the three independent experiments per method. B. Reduction of high-resolution data, generated by <i>xCELLigence</i>, to a low resolution comparable with data from conventional assays. The example shows migration (left) and invasion (<i>right</i>) of MDA-MB-231 cells through two densities of Matrigel. The ten time points in the Transwell method (<i>black arrows</i>) were selected from the <i>xCELLigence</i> plots (<i>grey</i> and <i>blue</i>) to reconstruct a low-resolution graph (<i>black</i>), directly comparable to the Transwell data. An identical approach was applied for all other processes studied.</p

Matrigel surface densities corresponding with degree of dilution for a fixed volume of 20 µL.

*<p>Matrigel (Basement Membrane Matrix, growth factor reduced, BD Biosciences) delivered as a ±13.55 µg/µL stock.</p

Time-dependent random migration profile of MDA-MB-231 and A549.

<p>Comparison of random migration signals (negative control – SF) between three quantitation methods: pixel area calculation – <i>black</i>, OD - <i>red</i>, xCELLigence - <i>green</i>. A likelihood ratio test revealed a significant difference in slope between area calculation and OD (p<0.001) and area calculation and <i>xCELLigence</i> (p<0.001) for both cell lines and OD and <i>xCELLigence</i> (p<0.001) for MDA-MB-231 only. OD and <i>xCELLigence</i> slopes did not differ significantly (p = 0.22) for A549 cells.</p

Conventional Transwell design for detection of time-dependent cell migration.

<p>A. A Transwell setup consists of an upper chamber (<i>insert</i>) that is placed onto a lower chamber (<i>well</i>). The insert contains a microporous membrane (8 µm pores) allowing passage of tumor cells. After a period of serum starvation a serum-free cell suspension is seeded in the insert and exposed to medium containing potential chemoattractants (by default: medium+FBS). During incubation at 37°C and 5% CO₂, cells migrate toward the bottom side of the membrane. B. Experimental design to assess time-dependent migratory behavior of cultured cells. Both migration toward FBS-containing medium and baseline migration (toward SF medium, no chemoattraction) as a negative control were included. Two times two 24-well Transwell plates were used to examine migration to FBS (positive control – <i>top row</i>) and baseline migration (negative control – <i>bottom row</i>). At ten time points during a 24-hour incubation period inserts were fixed and stained in duplicate. Two inserts containing cell-free media (grey fill) have been included throughout the experiment and fixed and stained after 12 hours incubation to assess background absorption in optical density (OD) measurements. In addition, to exclude influence of inter-plate variability on observed migration rates, each plate contained duplicate two-hour control inserts.</p

<i>xCELLigence</i> RTCA: impedance-based detection of cell viability and motility.

<p>Interdigitated gold microelectrodes on the well bottom (viability – E-plate) or on the bottom side of a filter membrane (motility – CIM-plate 16) detect impedance changes, caused by the presence of cells and expressed as a Cell Index. This detection method is proportional to both cell number (<i>left</i> and <i>above</i>) and morphology as increased cell spreading is reflected by a higher Cell Index value (<i>right</i>). When starting an experiment, a baseline Cell Index value is recorded in medium only before cell addition.</p