This application note demonstrates a method to measure cell migration in the Oris™ Cell Migration Assay by the use of ImageJ analysis software for counting cells. 16 hour migration on all surfaces p<0.005 statistical difference in cell migration between surfaces (two-sample t-test). p<0.001 statistical difference between control vs. Data are represented as average cell number +\- SD from 8 wells for each condition. (A) MDA-MB-231 and (B) HT-1080 cell migration on three surfaces (Tissue Culture Treated, Collagen I, or Fibronectin). Quantitation of Cell Number using ImageJ. Furthermore, this method of analysis yielded statistical differences in the migration of model cell lines on all three plate coatings (i.e., MDA-MB-231 migration on Collagen I versus Fibronectin).įigure 3. Both MDA-MB-231 and HT-1080 cells exhibited the most robust migration on Collagen I. Scale bar = 500 micrometers.įigure 3 shows the average number of MDA-MB-231 (3A) and HT-1080 (3B) cells that migrated into the Detection Zone when seeded on Tissue Culture treated, Collagen I coated, and Fibronectin coated wells. (M-P) Merged images of DAPI and particle analysis drawings. (I-L) Particle analysis drawings of objects in the ROI. (E-H), Fluorescent images of DAPI-labeled cells with a 2-mm circular ROI (red circle) defining the region for particle analysis.
(A-D) Phase images of migration control (A) and MDA-MB-231 cell migration on Tissue Culture Treated (B), Collage I (C), and Fibronectin (D) 16 hours after removal of stoppers from Oris™ Cell Migration Assay. ImageJ Analysis of Cell Migration by Counting Cell Number in the Detection Zone. Differences in MDA-MB-231 cell migration into the Detection Zone (ROI) were highlighted by overlaying the particle analysis drawing on the original fluorescent image (Figure 2M-P).įigure 2. Performing the particle analysis function in ImageJ yielded drawings of detected objects that were counted within the circular ROI (Figure 2I-L). Using ImageJ, DAPI-labeled cells were counted by creating a 2mm circular region-of-interest (ROI) similar in size to the initial Detection Zone (Figure 2E-H). Phase images of cells acquired immediately after stopper removal (migration control) and 16 hours after stopper removal demonstrate differences in cell migration depending upon whether cells were seeded on a Tissue Culture Treated surface, a Collagen I coated surface, or a Fibronectin coated surface (Figure 2A-D). MDA-MB-231 cells exhibited varying degrees of migration into the Detection Zone dependent upon the surface coating of the well (Figure 2). In this application note, MDA-MB-231 and HT-1080 cell migration on three surfaces (Tissue Culture Treated, Collagen I, or Fibronectin Oris™ Cell Migation Assay – TriCoated), was assessed by counting the number of cells in the Detection Zone using ImageJ. Additional information regarding the use of ImageJ for particle analysis can be found at ). The number of nuclei for each condition was averaged from 8 wells.
#COUNTING CELLS IN IMAGEJ WINDOWS#
The cell counts from the Summary Window (i.e., counts) were exported into Windows Excel for statistical analysis. “Summary” and “Exclude on Edges” were checked for “Show Masks” was selected to display a drawing of Particle size were 1 pixels2, respectively. Number of nuclei contained in the ROI was quantified using the menu command Analyze–>Īnalyze particles. The ROI was centered over the Detection Zone within each well. In the Specify window, “width” and “height” were set at 2mm, and the “oval” box Using the menu command Edit–> Selection–> Specify. Measuring 2mm in diameter (the same diameter as the tip of the stopper) was made Using the binary image, a circular region-of-interest (ROI) Overlapping nuclei were separated by performing a Watershed segmentation
Window, the thresholded image was converted to a binary image.
Threshold was set for each grayscale image ( Image–>Īdjust–> Threshold). Images were acquired using a 5X objective onĪ Zeiss Axiovert 200 inverted microscope equipped with a CCD camera.Ĭell migration into the Detection Zone was measured byĬounting cell number using ImageJ analysis software (version 1.42l). Afterġ6 hours, cells were fixed with 0.25% glutaraldehyde and cell nuclei were Tissue Culture Treated, Collagen I coated, or Fibronectin coated wells. MDA-MB-231 breast epithelial cells and HT-1080 fibrosarcomaĬells were cultured on an Oris™ Cell Migration Assay – TriCoated plate having
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