Figure 1.
xCELLigence RTCA: impedance-based detection of cell viability and motility.
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 (left and above) and morphology as increased cell spreading is reflected by a higher Cell Index value (right). When starting an experiment, a baseline Cell Index value is recorded in medium only before cell addition.
Figure 2.
Time-dependent proliferation and cytotoxicity profiles of MDA-MB-231 and A549.
A. Proliferation curves of MDA-MB-231 cells as generated by xCELLigence RTCA (red) and SRB (black) for different seeding densities of 100 (top left), 500 (bottom left), 1000 (top right) and 2000 cells/cm2 (bottom right) during a ten-day incubation. B. Same as (A) for A549 cells. All graphs represent results from three independent experiments ± SD. C. Cytotoxicity profiles relating to 72 hours of exposure to paclitaxel (0–100 nM). Cells were allowed to attach and propagate during 24 hours prior to start of treatment. Toxicity data from xCELLigence RTCA were derived from normalized plots. All graphs represent results from three independent experiments ± SD.
Figure 3.
Conventional Transwell design for detection of time-dependent cell migration.
A. A Transwell setup consists of an upper chamber (insert) that is placed onto a lower chamber (well). 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 – top row) and baseline migration (negative control – bottom row). 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.
Figure 4.
Time-dependent migratory pattern of MDA-MB-231 and A549.
A. MDA-MB-231 (left) and A549 (right) cell migration profiles, detected by Transwell experiments (black) and xCELLigence (red). Graphs represent scaled signals (0–1) of net chemoattraction after subtraction of the random migration signal (empty squares 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 (left panel) were normalized to a (0–1) scale (middle panel) through division of all data by the maximum value obtained in three independent experiments. Subsequently, random migration (SF) signals (triangle markers) were subtracted from the positive (FBS) control counterparts (circle markers) per experiment to obtain a pure chemotactic signal (right panel). Example shown is the migratory pattern of MDA-MB-231 cells estimated by pixel area calculation in three experiments (exp 1 - red, exp 2 - green, exp 3 - black). 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 (top row) and 16 hours (bottom row) of incubation. D. Migratory behavior of MDA-MB-231 cells toward medium+FBS (positive control – filled squares) and background migration (empty squares) as detected by conventional Transwell experiments at ten time points spread over 24 hours of incubation. Area calculation (left) of stained cells and optical density (OD – middle) were compared to the xCELLigence migration pattern, reconstructed from the original high-resolution plot by extrapolating data from the corresponding time points (right). 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.
Figure 5.
Time-dependent random migration profile of MDA-MB-231 and A549.
Comparison of random migration signals (negative control – SF) between three quantitation methods: pixel area calculation – black, OD - red, xCELLigence - green. A likelihood ratio test revealed a significant difference in slope between area calculation and OD (p<0.001) and area calculation and xCELLigence (p<0.001) for both cell lines and OD and xCELLigence (p<0.001) for MDA-MB-231 only. OD and xCELLigence slopes did not differ significantly (p = 0.22) for A549 cells.
Figure 6.
Time-dependent invasion profile of MDA-MB-231.
A. Experimental design to quantify MDA-MB-231 Matrigel invasion. Two times two 24-well Transwell plates were used to examine invasion to FBS through a 20% (v/v) (top row) and 7.7% (v/v) Matrigel layer (bottom row) after 24 hours of serum starvation. At ten time points during a 48-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 24 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 24-hour control inserts. B. MDA-MB-231 dynamic cell invasion profiles, generated by Transwell experiments (black) and xCELLigence (red). Graphs represent normalized signals (scaled values 0–1) of invasion through 20% (open circles), 10% (open squares), 7.7% (filled circles) and 3.3% (filled squares) to medium+10% FBS with associated Spearman's rank correlation coefficients (Rho). All results are from three independent duplicate experiments with SD. C. Sequential pictures showing invasive MDA-MB-231 cells at the indicated time points during a 48-hour incubation on Transwells coated with 20% (top row) and 7.7% Matrigel (bottom row). Pictures (obj. 2.5×) show cells fixed and stained in 20% methanol/0.1% crystal violet.
Table 1.
Matrigel surface densities corresponding with degree of dilution for a fixed volume of 20 µL.
Figure 7.
Prestatistical data processing.
A. Schematic depiction of processing kinetic data generated by SRB, xCELLigence and Transwells. Raw data with high time resolution (filled and empty circles), resulting from independent xCELLigence experiments (1, 2, 3 and grey arrows) are reduced to a lower time resolution by selecting only the data points corresponding with the time points of endpoint detection (filled circles 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 xCELLigence, to a low resolution comparable with data from conventional assays. The example shows migration (left) and invasion (right) of MDA-MB-231 cells through two densities of Matrigel. The ten time points in the Transwell method (black arrows) were selected from the xCELLigence plots (grey and blue) to reconstruct a low-resolution graph (black), directly comparable to the Transwell data. An identical approach was applied for all other processes studied.