The overall goal of this experiment is to quantify endothelial cell tube formation dependence on angiogenic chemokines using two different extracellular matrices that contain components which associate with various angiogenic molecules. This method can answer key questions at the cancer angiogenesis field, such as, why does angiogenesis support by alternate pathways using alterations in the extracellular matrix? Main advantages of this technique is that it is quantifiable and uses fewer variables.
Demonstrating the procedure will be Miss Donghong Ju, a research associate in the laboratory. To begin this procedure, thaw either GFR extracellular matrix, or normal extracellular matrix, overnight at four degrees Celsius. Keep the 96-well culture plate on ice and add 50 microliters of chilled extracellular matrix per well using pre-cooled pipette tips.
Prepare triplicates of five wells containing only the normal extracellular matrix, followed by triplicates of five wells containing only GFR extracellular matrix. Then, incubate the plate at 37 degrees Celsius for 30 minutes. After 30 minutes, wash the HUVEC once with PBS, without calcium and magnesium, and add one milliliter of 05%of Trypsin EDTA.
Subsequently, incubate the cells at 37 degrees Celsius for one to five minutes, and check them every minute until most cells round up. Then, dislodge the cells by tapping the flask. Afterward, add five milliliters of Basal medium with one percent FBS to the flask.
Transfer the cells to a 15-milliliter tube. Then, centrifuge the cells at 200 times G for five minutes. After five minutes, discard the supernatant and re-suspend the cells with two milliliters of Basal medium.
Count the cells using a hemocytometer and adjust the cell concentration to two to three times 10 to the fifth cells per milliliter. Then, prepare 100 microliters of five different 10X concentrations of CXCR2 inhibitor in Basal medium. Pipette 300 microliters of the HUVEC suspension into each of the five individual microcentrifuge tubes.
Subsequently, add 33 microliters of the 10X inhibitor of each concentration into the tubes containing HUVEC and vortex them. Next, pipette 50 microliters of each of the cell suspensions into individual wells of the extracellular matrix. Plate each suspension in triplicate, and incubate for four to 16 hours at 37 degrees Celsius.
Afterward, take four pictures per well using an inverted microscope camera. In this procedure, prepare the extracellular matrix and plates according to the same specifications as the tube formation assay. Afterward, prepare the HUVEC suspension and aliquot into a 96-well plate containing the extracellular matrix at 50 microliters per well.
Following this, grow the cells on the extracellular matrix for four hours at 37 degrees Celsius. Then, take images before the initiation of drug treatment. Next, prepare five different two X concentrations of the CXCR2 inhibitor in Basal medium.
Add 50 microliters of CXCR2 inhibitor of each concentration to each of the 96 wells containing HUVEC. Afterward, incubate the plate for another two to 12 hours at 37 degrees Celsius. Then, take four images of each well using an inverted microscope camera.
To evaluate tube formation captured in the images, measure the total tube length in four random microscopic fields using the microscope camera software. To do so, open the image in the microscope camera software and click Annotations and Measurements. Under the Length section, select the simple line tool.
Click on the image, then draw a line along the length of the tube, then right-click to perform calculation. Afterward, repeat the procedure for all the tube lines in the image by selecting the simple line tool. Click on the image, draw a line along the length of the tube, then right-click.
The software will record the length of each line and display the data on the screen. After measuring every tube in the picture, click Export, then choose Length to Spreadsheet to export the length data to a spreadsheet. Add all of the tube lengths from the spreadsheet to get the total tube length.
Next, calculate and record the average length of the tubes. In this procedure, culture and perform a tube formation assay in the 12-well plate. Incubate the cells for four to 16 hours in order to form the tubes.
Afterward, aspirate the cell culture medium and rinse the cells with one milliliter of cold 1XPBS without calcium and magnesium. Subsequently, add one milliliter of ice-cold PBS buffer with 2.5 millimolar EDTA onto the culture and keep it on ice for 10 minutes. After 10 minutes, dislodge the cells and extracellular matrix mixture from the dish using a 1, 000-microliter pipette tip with the tip cut off.
Then, transfer the cells to a cold, 15-milliliter tube. Wash the well with four milliliters of ice-cold PBS and put into the same tube. Next, put the tube on ice for one to four hours and invert the tube a few times until the extracellular matrix is dissolved.
After that, centrifuge it for 10 minutes at zero degrees Celsius. Then, re-suspend the cell pallet with one milliliter of cold PBS in a 1.5 milliliter tube on ice. Centrifuge it again for five minutes at zero degrees Celsius.
Then, dispose of the supernatant and store the cells at 80 degrees Celsius. These two images show successful endothelial tube formation on the normal extracellular matrix and GFR extracellular matrix. The interconnected network of tubes clearly shows that the growth of tubes is healthy in these HUVEC.
These two images show the inhibition of tube formation by a CXCR2 inhibitor, SB225002, at 5.6 micromolar on the normal extracellular matrix and GFR extracellular matrix. The isolated clumps of HUVEC seen in this image show that the cells were not able to form the necessary tubes to connect to each other. This graph shows that the HUVEC response to the CXCR2 inhibitor on GFR extracellular matrix is dose-dependent.
Following the procedure, other methods like QRTPCR, or RNA sequencing can be performed using the prepared cells. Data like average tube length, total number of tubes, or total number of branch points, can be obtained using images in order to characterize the angiogenesis process under experimental conditions.
