The cell stretching project focuses on simulating cell deformation through the use of the Controlled Axonal Injury (CAI) Device. Damaged cell samples are then studied with the use of fluorescence imaging to determine the extent of injury in proportion loading.
Controlled Axonal Injury (CAI) Device
The first step in analyzing the neurons' damage thresholds is to perform a controlled deformation on the cells. Within this line of research, deformation has been achieved in two ways, the most recent of which is through use of the CAI (Cultured Axonal Injury) System. It introduces compressed air into an airtight chamber to deform the transparent membrane, thus stretching the cell. The Flexcell system has also been used and more can be read about it here.
The strain and strain rates were calibrated using high-speed ARAMIS. Typically the frame rate of 5000 fps was used to capture the PDMS membrane deformation caused by pressure pulses with the nominal time of 10-50 ms.
The video shows a set of representative strain calibration curves captured and analyzed with high-speed ARAMIS. The plot shows strain magnitude as a function of time in 6 points indicated with different colors in the inset. The inset presents the deformation of the PDMS membrane, the calculated strain map and its evolution with time.
Viability of Non-Differentiated Neuroblastoma SH-SY5Y Cells
|Calcein-AM (green), the acetomethoxy ester form of calcein, can penetrate the intact membrane of healthy cells. Intracellular esterases cleave the ester group, which makes the calcein strongly fluorescent. Dead cells do not have functional esterases and can’t be labeled with this method. Ethidium homodimer-1 (red), a membrane impermeant fluorescent dye is used to stain dead cells exclusively. The viable cells with compromised membrane will be stained green and red, and these cells are classified as injured. Using CompuCyte LSC, we evaluated populations of cells subjected to different levels of strain and strain rates. We demonstrated the combination of classical Live-Dead assay and LSC capabilities enabled us to evaluate in single experiment the population of injured cells. These measurements were conducted on samples of at least 8000 adherent cells over the area of 3 cm2 per specimen.|
|Fluorescence imaging will be used to study the disruption of ionic homeostasis and plasma membrane injury. The acute influx of Ca2+ accounts for many cellular responses including cell death via apoptosis and necrosis (Raghupathi et al. 2004). Ca2+ dye and fluorescence microscopy will be especially useful in the study of dynamic cellular Ca2+ variation.|