Valarmathi - Video - 1 - MSCs-Control-24 Hours-10X Ph.AVI

Live Cell Imaging and Analysis

Image Acquisition

     Live cell imaging and Image acquisition set up were exactly followed as per our previously published protocols (Fuseler and Valarmathi, 2012).

 

     “Following creation of the wound area in the MSCs monolayer cultures, the MatTek dishes were immediately mounted on the stage of a Zeiss Axiovert 200M inverted microscope in a Zeiss M200 humidified incubator chamber, and maintained at 37^oC and 5% CO₂ during the course of observations and the collection of images. An appropriate area of the margin of the wound was selected for imaging. Images were collected by time-lapse microscopy using phase contrast optics (10X, N.A. 0.25 PH-1 Achroplan objective) and a CCD camera (Hamamatsu ORCA-ER) controlled by Kinetic Imaging Image Acquisition Software (AQM Advance-6) software at a rate of 10 frames per hour for a total period of 24 hours. The incident light during exposure of the image was filtered with 546 nm green interference filter, and exposure times were maintained constant at 23.15 ms for all kinetic cell migration experiments,” (Fuseler and Valarmathi, 2012).

 

Measurement of MSCs Cellular Movements and Displacements

     The effect of exogenous NO on directed cellular movement was determined by measurement of the displacement of individual MSCs into the wound region. The changes in the displacement of individual MSCs were determined by frame-by-frame analysis with the AQM Advance-6 software, and were expressed as the mean of the final displacement of the MSCs from the wound margin at 24 hours. On an average, 50 cells were measured for each experimental group treated with SNAP (SN-1 and SN-2) and their corresponding control groups.