The overall goal of this procedure is to culture human umbilical vein endothelial cells and to analyze cytoskeletal dynamics and cell migration using live cell time lapse imaging. Methods described in this video can help answer key questions in the field of cell, molecular, and mechanobiology. For example, this protocol can be used to identify how cell shape and motility are modulated by engagement of different ECMs.
Visual demonstration is critical because the sample preparation steps are difficult to perform. They require precise handling of the cells in a timely manner throughout the process. Though this method can provide insight into endothelial cell motility during angiogenesis, it can also be applied to microscopic investigations of virtually any adherent cell model system.
Using forceps, pick up a coverslip from a 100%ethanol storage jar and pass it through a Bunsen burner flame to sterilize it. Then place it in a 35 millimeter petri dish. Prepare one coverslip for each sample.
To a 1.5 milliliter microcentrifuge tube add 10 microliters of one milligram per milliliter fibronectin stock solution and 990 microliters of PBS. Then pipette 250 microliters of the solution onto each coverslip. Spread the solution evenly to cover the surface.
Incubate the coverslips for a minimum of three hours at 37 degrees Celsius. Before use, rinse each coverslip three times with two milliliters of PBS. Using a hemocytometer, determine the number of cultured HUVECs and transfer a volume containing 100, 000 cells per coverslip for non-migration experiments, or 500, 000 cells per coverslip for migration experiments to a microcentrifuge tube.
Spin the cells at 1, 400 times times G for four minutes to pellet them. After the spin, for each condition to be tested, resuspend up to one million cells in 100 microliters of electroporation buffer and combine them with one microgram of cDNA. Transfer the cells to an electroporation cuvette.
Transfect the cells and DNA mixture using the program designated for HUVEC electroporation. Next, rescue the transfected cells by adding 500 microliters of complete endothelial basal medium. Then, plate the appropriate cell volume onto fibronectin-coated coverslips.
Incubate the cells at 37 degrees Celsius for three to four hours to allow time for the expression of the cDNA constructs. In preparation for imaging, take two 2.5 centimeter by 0.65 centimeter strips of double sided tape and secure them horizontally along the top and bottom edge of a clean glass slide. Note that it is important to allow a small amount a space between the tape and the slide edge for sealing the chamber.
Mount the coverslip cell-side down such that the two edges of the coverslip rest on the tape. Use forceps to press down gently to secure the coverslip. Using a micropipette, add 40 microliters of imaging medium between the coverslip and slide.
Ensure that the space between the coverslip and the slide is completely filled with imaging medium. Blot any excess medium with a paper wipe if necessary. Seal all of the slide edges with warm VALAP.
Check for leakages by pushing gently on the glass coverslip. Place the mounted and sealed coverslip onto a microscope stage pre-warmed to 37 degrees Celsius. Using a spinning disk confocal microscope, capture time lapse image sequences, blue fluorescent protein images, and DIC images.
Analyze the microtubule dynamics and co-localization as described in the accompanying document. To perform a cell migration assay, after the electroporation step, rescue the transfected cells by adding 80 microliters of complete endothelial basal medium. Then, pipette the entire 80 microliter sample as a single drop onto the center of a dried fibronectin-coated coverslip.
Do not spread the drop. Drying the coverslip is necessary to prevent the 80 microliter drop from separating upon contact with the coverslip. This approach allows for HUVECs to be concentrated within a small area of the coverslip and thereby promotes the rapid formation of a confluent monolayer of cells.
Incubate the coverslip at 37 degrees Celsius for three to four hours to allow time for the cells to adhere into a confluent monolayer. Then rinse the slides two times in complete endothelial basal medium to remove any cells that have not attached. To create a wound edge on the HUVEC monolayer, use forceps to gently hold the coverslip in place and firmly drag a razor blade across the center of the cell monolayer.
Allow the cells to recover in a 37 degree Celsius incubator for three hours. Then assemble and mount the coverslips for imaging as before. Acquire phase contrast images at 10 minute intervals for 12 hours using a 10X 0.45 numerical aperture phase objective and a 0.52 numerical aperture long working distance condenser.
Use the ND Acquisition panel of the image acquisition software program. Finally, quantify HUVEC branching and migration as described in the accompanying document. HUVECs were transfected to express mApple-labeled-EB3, a microtubule end-binding protein and time lapse images were captured.
Raw images of EB3 are shown on the left. Blue EB3 comets tracked by plusTipTracker software are shown on the right. Comets detected within each frame were linked based on their change in position from frame to frame to generate EB3 tracks and a visual EB3 track overlay, as shown here.
Slow, short-lived microtubule growth is shown in red, slow, long-lived microtubule growth is shown in green, fast, short-lived microtubule growth is shown in yellow, and fast, long-lived microtubule growth is shown in blue. To analyze endothelial cell branching, a DIC and a fluoresent image of an individual HUVEC was captured at 60 times magnification. A boundary was hand-drawn around the cell edge in the fluorescent image.
Branch origins were then defined by drawing a straight line across the branch and through the greatest angle of curvature on either side of the branch. Branches that were longer than they were wide and minimally 10 microns in length were counted and length was measured from the branch origin to the distal tip of the branch. To assess endothelial cell wound edge migration, HUVECs were cultured at high density and live cell time lapse imaging was performed following wounding as described in this video.
As shown here, wound-edge endothelial cells migrated directionally into the wound area. This movement was analyzed using the nucleolus as a fiduciary marker to track migration velocity, persistence, and distance. After watching this video you should have a good understanding of how to culture, transfect, and prepare HUVECs for either cell shape and cytoskeletal dynamics imaging experiments or for wound-edge cell migration studies.
Following this procedure, other methods like polyacrylamide or collagen conjugation can be performed in order to answer additional questions related to cell engagement of modified ECMs. Thanks for watching, and good luck with your experiments.
