Introduction
Cell migration, the movement of cells from one location to another, is a critical component of both normal and abnormal biological processes. The importance of cell migration in diverse biological processes has led to the development of systems for studying the mechanisms responsible in an effort to identify therapeutic components capable of either promoting (wound healing) or inhibiting (tumor formation) cell migration.
This study focuses upon the feasibility of using the SpectraMax® MiniMax™ 300 Imaging Cytometer, an optional upgrade for the SpectraMax® i3 Multi-Mode Microplate Reader, to visualize and analyze cell migration using the Oris™ Pro Cell Migration Assay from Platypus Technologies. This assay utilizes a non-toxic biocompatible gel that dissolves and creates a cell-free detection zone in the center of each well, allowing for the study of the cell migration mechanisms involved as cells move into the detection zone.
Materials and methods
Cell culture and assay
HT1080 cells were seeded into either 96-well (20,000 cells/well) or 384-well (8,000 cells/well) Corning black, clearbottom plates provided with the Oris Pro Cell Migration Assay. Here the Collagen I Coated version was used (Platypus Technologies cat. #PROCMACC1). The assay uses a non-toxic biocompatible gel to create a cell-free zone on cell culture surfaces.
After seeding, cells were placed in a humidified chamber (37°C; 5% CO2) for approximately four hours to permit cell adherence. Subsequently, fresh media was added with either the test compound cytochalasin D, which is a cell-permeable mycotoxin known to disrupt cell migration, or a 0.1% DMSO vehicle control.
After 48 hours, the plates were removed from the incubator. The MiniMax cytometer was used to acquire transmitted light images of each well for later analysis using Molecular Devices StainFree™ Cell Detection Technology.
Cells were stained with CellTracker™ Green CMFDA Dye (Thermo cat. # C2925), following the manufacturer’s instructions, and fluorescent images were acquired using the green fluorescent channel of the MiniMax cytometer. After images were acquired, a fluorescence endpoint read (excitation 472 nm, emission 526 nm) was taken to determine whether the fluorescence detection mode of the SpectraMax® i3 reader could be used to detect cell migration.
The amount of cell migration in each well of the microplate was analyzed by specifying a region of interest in the acquired images that corresponded to the detection zone created by the dissolved biocompatible gel. SoftMax® Pro Software was then used to calculate the amount of cell migration in transmitted light images using the StainFree technology. For cells stained with CellTracker Green, a cell detection algorithm based on fluorescence intensity in SoftMax Pro was used (Figure 1).
Figure 1. Schematic of Oris Pro Cell Migration Assay. This assay uses a non-toxic biocompatible gel to form a cell-free zone in the center of the well. After cells are seeded, the biocompatible gel dissolves, allowing cells to migrate into the detection zone in the center of the well. For images acquired with the MiniMax cytometer, a region of interest corresponding to the detection zone is specified, and the level of cell migration (area covered by cells) is analyzed.
MiniMax cytometer and SpectraMax i3 reader parameters
Cell migration was evaluated with the MiniMax cytometer by analyzing the portion of the images representing the detection zone of each well. For each well in 96-well or 384-well plates, either a single site per well or four sites per well were imaged. When four sites were imaged, SoftMax Pro Software automatically created a tiled composite image for each well. In addition to image analysis, the fluorescence detection mode of the SpectraMax i3 reader was also used to measure cell migration into the detection zone (Table 1).
| SpectraMax i3 – Endpoint | MiniMax cytometer – Imaging | |
|---|---|---|
| Plate types | 96- and 384-wells | 96- and 384-wells |
| Read mode | Fluorescence | Camera |
| Read type | Endpoint with bottom read | Fluorescence or transmitted light |
| Wavelengths/bandwidths | Ex: 472 nm/15 nm Em: 526 nm/25 nm |
Ex 456/Em 541 or TL channel |
| Reads per well | 1 read, 6 flashes per read | 1 site per well or 4 sites per well |
Table 1. Instrument settings for cell migration assay.
Data analysis
For the MiniMax cytometer, one or four sites were imaged from the center of each well. SoftMax Pro Software was used to specify a region of interest in the images corresponding to the cell-free detection zone created by dissolution of the biocompatible gel. Cells that migrated into the detection zone were identified by the software. Results were expressed as a percentage of the total area covered by the migrated cells versus the total imaged area.
For the SpectraMax i3 reader, fluorescence intensity measurements were acquired using a bottom-read endpoint read type. Data were graphed using a 4-parameter curve fit, and cytochalasin D IC50 values were calculated and reported by SoftMax Pro Software.
Results
The dose-dependent effects of cytochalasin D on the inhibition of cell migration were measured by exposing the cells to either 0.1% DMSO or increasing concentrations of cytochalasin D.
Both transmitted light and fluorescent. CellTracker Green-stained cell images were acquired using the MiniMax cytometer to visually ascertain the level of cell migration in each well. Either a single site or four sites per well were imaged to determine the optimal number of imaged sites needed to accurately measure cell migration.
In 96-well plates, a clear visual difference between the cytochalasin D and vehicle control wells could be seen in the transmitted light and fluorescent images for both the one site (Figure 2) and four sites (Figure 3) per well acquisitions. When the images were analyzed, cytochalasin D displayed a strong concentrationdependent effect on the level of cell migration into the detection zone (Figure 4).
Figure 2. Cytochalasin D has a visible inhibitory effect on the level of cell migration into cell-free detection zone in 96-well plates. The cell-free detection zone of each well was surveyed by imaging a single site per well for both transmitted light (A and C) and green fluorescence (CellTracker Greenstained cells; B and D). Wells were exposed to either 0.1% DMSO (A and B) or 2 μM cytochalasin D (C and D). In wells exposed to the DMSO control, many cells migrated into the detection zone. With the 2 μM cytochalasin D, very few cells migrated into the detection zone.
Figure 3. Cell migration imaged using four sites per well. The cell-free detection zone of each well was surveyed by capturing a four-image montage in both transmitted light (A and C) and Green Channel fluorescence (CellTracker Green-stained cells; B and D). Wells were exposed to either 0.1% DMSO (A and B) or 2 μM Cytochalasin D (C and D). Similar to images of a single site per well, the DMSO control showed much higher numbers of migrated cells than did the cytochalasin-treated wells.
Figure 4. Cytochalasin D displays a dose-dependent inhibition on cell migration in both transmitted light and CellTracker Green-stained images from 96-well plates. Wells were exposed to various concentrations of Cytochalasin D and either transmitted light images (black plot) or CellTracker Green-stained images (green plot) of the well center (one or four sites per well) were analyzed to determine the level of cell migration into the detection zone. The level of cell migration into the detection zone was analyzed as percent area covered by cells in a region of interest in the center of each well.
The dose-response effect of cytochalasin D on the inhibition of cell migration was then assayed in 384- well plates to determine the potential effectiveness of the Oris Pro Cell Migration Assay in high-throughput environments. Similar to the images from 96-well plates, a noticeable difference in cell migration into the detection zone could be seen between the cytochalasin D and DMSO control wells in both the transmitted light and green fluorescent channel images (images not shown). No qualitative difference in cell migration could be seen between the one site and four sites per well acquisitions. When the images were analyzed, cytochalasin D again displayed a strong concentration-dependent effect on the level of cell migration into the detection zone (Figure 5).
Figure 5. Cytochalasin D displays a dose-dependent inhibition on cell migration in both transmitted light and CellTracker Green stained images from 384-well plates. Wells were exposed to various concentrations of Cytochalasin D and either transmitted light images (black plot) or CellTracker Green-stained images (green plot) of the well center (one or four sites per well) were captured to determine the level of cell migration into the detection zone. The level of cell migration into the detection zone was analyzed as percent area covered by cells in a region of interest in the center of each well.
In addition to image analysis, fluorescence intensity of the CellTracker Green-stained cells was measured using the fluorescence detection mode of the SpectraMax i3 reader (Figure 6). In both 96-well and 384-well plate formats, increasing concentrations of cytochalasin D resulted in a significant decrease in fluorescence intensity that parallels the decrease in cell migration observed in the images acquired using the MiniMax cytometer. These results confirm that the SpectraMax i3 reader is sensitive enough to detect cell migration using the Oris Pro Cell Migration Assay plates. IC50 values and Z' factors were calculated in SoftMax Pro Software, and the results are summarized in Table 2. Similar results were observed for StainFree cell analysis, fluorescence image analysis, and fluorescence plate reads.
Figure 6. Cells stained with CellTracker Green show a decrease in fluorescence when exposed to increasing levels of Cytochalasin D as measured by the SpectraMax i3 reader. Cells in 96-well plates (left) or 384-well plates (right) were exposed to increasing concentrations of cytochalasin D, and CellTracker Green fluorescence was measured using the endpoint read type. The dose-dependent effect of cytochalasin D on the decrease in fluorescence intensity corresponds to an inhibition of cytochalasin D on cell migration.
Summary
Using the transmitted light and green fluorescence imaging capabilities of the MiniMax cytometer, cytochalasin D was clearly shown to inhibit cell migration in Oris Pro Cell Migration Assay plates. Similar inhibitory effects were seen in both 96- and 384-well assays. In both 96- and 384-well plate formats, the inhibitory effect of cytochalasin D on cell migration could be measured whether one site or four sites were imaged per well. These results indicate that the Oris Pro Cell Migration Assay can be used to accurately measure cell migration from a single image per well, enabling more rapid data acquisition for an entire 96-well plate or higher-throughput 384-well plate format.
Cytochalasin D inhibition of cell migration could also be measured using the fluorescence detection mode of the SpectraMax i3 reader, although standard deviations were higher than with image analysis. These results offer users the option to study cell migration on the SpectraMax i3 reader using its onboard fluorescence detection capability.
The SpectraMax i3 Multi-Mode Microplate Detection Platform is ideal for studying cell migration with the Oris Pro Cell Migration Assay from Platypus Technologies. Patentpending Spectral Fusion™ illumination gives the SpectraMax i3 reader optimal sensitivity and extended signal range to handle demanding assays such as cell migration. The platform can be upgraded with the MiniMax cytometer, which provides the flexibility of StainFree or fluorescence image analysis, with even larger signal windows.
| MiniMax CYTOMETER | SpectraMax i3 | ||||
|---|---|---|---|---|---|
| Transmitted light | Green channel fluorescence | Endpoint read Ex. 472 nm Em. 522 nm |
|||
| 1 image/well | 4 images/well | 1 image/well | 4 images/well | ||
| 96 well | 0.130 | 0.138 | 0.139 | 0.147 | 0.060 |
| 384 well | 0.129 | 0.138 | 0.106 | 0.109 | 0.142 |
Table 2. Summary of IC50 (nM) values obtained via different detection methods on the SpectraMax i3 platform. Cytochalasin D displays similar IC50 values across transmitted light images, Green-channel fluorescence images, and endpoint fluorescence reads.
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