Computational protocol: Direct expression of active human tissue inhibitors of metalloproteinases by periplasmic secretion in Escherichia coli

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Protocol publication

[…] For 3D imaging, the cells were trypsinized and suspended at a concentration of 600,000 cells/mL in a 2 mg/mL non-pepsin-treated rat collagen type I solution. 50 nM N-TIMP-1 and N-TIMP-2 were added to this solution prior to the addition of the collagen. A 125 μL volume of the solution was pipetted into a MatTek dish (MatTek Corporation, Ashland, MA, USA) and allowed to polymerize at room temperature for 45 min. After polymerization, 125 μL of imaging media was added to the top of the gel. To seal the chamber, a 5-mm thick pad of polydimethylsiloxane (PDMS) (Dow) punctured with an acupuncture needle was placed on top and sealed with food grade lubricant (McGlaughlin Oil Company, Columbus, OH). The acupuncture needle was then rotated one full rotation to align the collagen fibers and placed in a 37 °C incubator. After 24 h, the chambers were removed from the incubator and imaged every 2 min for 8 h. The images were analyzed using the MTrackJ plugin in ImageJ which allows the user to track the cells on an image-by-image basis. The plugin generates the x–y coordinates of the cell at each time point. Trajectories were analyzed using a custom Matlab script to calculate a migration speed. Directionality was determined using another custom Matlab script. The angle between the direction of the migration and a radial vector field originating from the needle was used to calculate directionality with the following equation:2DI=cos2θ Aspect ratio was calculated by measuring the length of a cell and dividing it by the width of that same cell. Analysis of cell morphology was completed by binning cell shape based on the shape created by the extensions after the cells had time to adhere to its environment: 9 h for 2D and 24 h for 3D. 95% confidence intervals were calculated using Matlab with a sample of at least 79 cells. A two tailed t test at p ≥ 0.05 was conducted to identify statistical differences between the N-TIMP conditions and that of the control. […]

Pipeline specifications

Software tools MTrackJ, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens, Escherichia coli
Diseases Arthritis, Neoplasms, Nervous System Diseases, Atherosclerosis