The goal of this procedure is to observe dynamic changes in the actin cytoskeleton of live endothelial cells. First transfect human umbilical vein endothelial cells with A GFP beta actin, plasmid, and culture to confluence on cover slips. Then position the cover slips into a live cell imaging chamber with a heated stage and an albumin physiological sort solution infusion.
The final step is data acquisition of a selected area of cells via a time-lapse image capture before and after treatment with a pharmacologic agent. Chime graph analysis with imaging software such as Image J can provide insights into local lamely podia protrusions, such as distance, time, and velocity of protrusions, as well as acting fiber movement over time. The main advantage of this technique over existing methods like immunofluorescence imaging, is that live cell imaging allows the incorporation of the dynamic nature of the act and cytoskeleton into studies of the mechanisms determining endothelial permeability.
Whereas fixed stain cells represent snapshots in time and provide low information of the dynamics of the act and cytoskeleton before and after treatment with an inflammatory mediator In a biological safety hood. Sterilized cover slips with 70%ethanol for two minutes and then air dry position. Each cover slip into a 10 centimeter culture plate and add 300 microliters of warm gelatin solution to the center without touching the edges of the cover slip After five minutes, aspirate the liquid.
Now harvest a confluent culture of human umbilical vein endothelial cells or VE with 0.25%tripsin EDTA. Count the cells and fill it by centrifugation. Aspirate off as much of the SUP natum media as possible for a transfection reaction with the VE nuclear effector kit.
Re suspend the cell pellets in basic nuclear effector solution and add 0.2 to two micrograms of GFP beta actin vector plasmid. Transfer 100 microliters of cell suspension to a nuclear effector qve. Tap the covered vete a few times to ensure there are no bubbles between the bottom of the Q vet and the transfection sample.
Place the Q VET into the nuclear effector two device and run program A 0 3 4 using a transfer pipette from the nuclear effector kit at a warmed quart of 500 microliters EGM two MV media to the qve. Then transfer the mix back to the micro fuge tube and incubate for 15 minutes. Gently mix the transfected VE suspension once and see 300 microliters of suspension onto a gelatin coated cover slip without touching the edges.
Then incubate for one to four hours. Inspect the transfected cells to confirm attachment to the cover slip. Then add 10 milliliters of E GM two MV media to the plate, and continue to incubate for GFP acting expression.
First, add an albumin physiological salt solution medium or a PSS to the gravity flow system and to connect to the inline heater. Now adjust the flow regulator to achieve a flow of approximately 40 milliliters per hour. Then stop the flow by closing the stop cock with a cotton tipped applicator.
Apply vacuum grease to the outer edge on the underside of the diamond bath. Now remove a transfected culture from the incubator. Gently lift the cover slip using forceps.
Then using a Kim wipe, soak up excess EGM two MV media on the bottom slide, positioning the cell's face up. Place the diamond bath over the cover slip to form a chamber. Quickly place the chamber into the stage heater.
Tighten the clamps over the chamber and pipette approximately one milliliter of EGM two MV media from the plate into the chamber. Now attach the vacuum hose to its holder on the left side of the chamber and the A PSS line from the inline heater into the channel on the right side of the chamber. Then turn on the vacuum and open the A PSS line to allow the bath perfusion over the transfected cells.
Also switch on the inline heater at 37 degrees Celsius. Next place the thermistor probe for the heated stage into the bath at the edge and switch on the stage heater at 37 degrees Celsius. To remove any remaining EGM two MV media and sort buildup, carefully wipe the underside of the cover slip using a Kim wipe Soaked with 70%ethanol, followed by a dry Kim wipe equilibrate the transfected HU cells for 30 minutes.
Lock the course. Focus on the selected area of culture cells for study. Set the camera exposure time between 0.5 and two seconds depending on the GFP Act in signal intensity in the cells.
Also set the interval and duration times for the experiment. Now with the room lights off, capture a single image to evaluate the settings. Capture the time lapse series monitoring the images as they they're required.
In a typical protocol, obtain 20 to 30 minutes of baseline images before adding a test agent, followed by 0.5 to four hours of additional image acquisition. Finally, as detailed in the accompanying text, evaluate lamely podia protrusion distance, persistence, and velocity by CH graph analysis. In a typical experiment, GFP actin is expressed in over 50%of the transfected VE in a random manner.
These images acquired once per minute captured GFP actin throughout the cytoplasm, as well as in filamentous structures and in local e meli podia protruding along the cell edge. The distance persistence and velocity of cell protrusions are quantified from time-lapse image sets. Using chm graph analysis, first, a single pixel line is drawn roughly perpendicular to the edge of the cell.
This region is extracted from each image of the time-lapse set to generate a montage of the region over time or chime graph here from a left to right perspective in the chime graph, protrusions are represented as upward movements in the edge of the cell. When a line is superimposed on the edge of one of these protrusions, the pixel data associated with that line can be collected to quantify protrusion dynamics. The total number of protrusions in a chm graph can be used to estimate the protrusion frequency.
This analysis depicts stress fiber movements formed near the cell periphery and move towards the cell center where they eventually disassembled. On the chio graph, the stress fibers appear as continuous lines in the cytoplasmic area, often moving down and to the right towards the cell center when the fibers are difficult to see in the original chio graph, the unsharp mask filter sharpens the image lines drawn on the stress fibers facilitate pixel data collection using the measure function. Alternatively lines drawn from the start to finish of the identified stress fiber, a mass average pixel data for the duration that the fiber was observed.
This presentation demonstrated how to analyze data from the time-lapse image set via graph analysis with software such as Image J.After watching this video, you should have a good understanding of how to study acton cytoskeleton dynamics in response to inflammatory mediators using live cell imaging of transfected endothelial cells.
