Quantitation of Endothelial Cell Adhesiveness In Vitro

The overall goal of this procedure is to quantify the adhesiveness of endothelial cells in vitro. This is accomplished by first preparing fibronectin coated cover slips in a 24 world cluster plate. Human coronary artery endothelial cells are then applied to the glass cover slips and left to incubate until a confluent endothelial cell monolayer is formed.

After administration of the desired treatment to the endothelial cells, fluorescently labeled monocytes added to the glass cover slips and incubated with the endothelial cell monolayer. The final step is the removal of unbound monocytes from the endothelial monolayer by a meticulous dunking and washing procedure of the cover slips. Ultimately, this method allows quantification of the percentage of an endothelial cell population that is adhesive after a particular treatment.

The main advantage of this technique is that it measures the actual number or percentage of adhesive endothelial cells as opposed to the overall stickiness of an endothelial mono layer as measured by all current methods. We first have the idea for this method when we attempted to measure endothelial cell ADIs using currently available methods and encountering unacceptably large variation in the results due to technical challenges inherent with these methods. Sterilized 12 millimeter diameter round glass cover slips by soaking them in 70%ethanol for at least 10 minutes with occasional agitation to ensure total exposure of all cover slips to the ethanol.

Then all the glass cover slips and ethanol into a sterile cell culture dish with a pair of sterile fine five B forceps. Pick up and place each individual glass cover slip into a well of a 24 well cluster plate. Take care to ensure that the cover slips are not touching the size of the well and are approximately in the middle of the, well leave the uncovered 24 world cluster plate with glass cover slips to dry in the flow cabinet.

This takes about 10 minutes. When the cover slips are dry, I pet 100 microliters of coating solution onto each glass cover slip so that the solution covers only the glass without pulling around the outside of the well. Place the covered 24 well cluster plate in a cell culture incubator for at least an hour.

Culture, human coronary artery endothelial cells in medium at 37 degrees Celsius and 5%carbon dioxide aspirate off the culture media and rinse the endothelial cell monolayer with 10 milliliters of Hank's balance salt solution or HBSS then aspirate off the HBSS at two milliliters of 0.5%trips in 0.2%EDTA solution and incubated room temperature for approximately five minutes when the endothelial cells can be dislodged by a tap to the side of the flask. At five milliliters of soybean trypsin inhibitor squirting the surface of the flask. To further dislodge the cells, transfer the cells into a 15 milliliter tube and centrifuge the tube at 200 times G for five minutes, aspirate off the supinate and resuspend the endothelial pellet in five milliliters of media.

Count the cells with a hemo cytometer and adjust the cell concentration to 50, 000 endothelial cells per milliliter of media. Dispense one milliliter of the cell suspension into each well of the 24 well plate containing coated glass cover slips incubate the cells overnight in a cell culture incubator at 37 degrees Celsius plus 5%carbon dioxide. The cells are ready for the desired treatment.

When a confluent monolayer is formed within three days transfer HL 60 cells that are cultured in RPMI is supplemented with 10%fetal calf serum into a 15 milliliter tube and centrifuge the cells at 200 times G for five minutes. Then remove the supinate and resuspend the cells in five milliliters of media without serum for fluorescence labeling of the HL 60 cells. Count and transfer the appropriate amount of cells into a 15 milliliter centrifuge tube.

Centrifuge the cells at 200 times G for five minutes. After removing the supinate, re suspend the cell pellet in cell tracker in our PMI media without serum. At a concentration of 1 million cells per milliliter, incubate the cells in the cell culture incubator for one hour following incubation, centrifuge the cells at 200 times G for five minutes and discard the supinate Reese.

Suspend the cell pal in media to a concentration of 2 million cells per milliliter. Add one milliliter of cell tracker labeled HL 60 cell suspension into each well of the 24 well cluster plate containing the endothelial cell monolayer and incubate the plate in a cell culture incubator of 37 degrees Celsius and 5%carbon dioxide for a chosen fixed period of time between one and three hours after the incubation period. Use a pair of fine five B forceps and pick up the glass cover slip from the well ripping only the outer edge of the cover slip both the cover slip and dab the edge of the cover.

Slip on a piece of tissue on the bench for three seconds. Taking care not to touch the cell covered surface of the cover slip with the tissue holding the cover slip firmly with a pair of forceps. Dunk the cover slip in and out of the HBSS five times after the fifth dunking.

HBSS dab the edge of the cover. Slip on the tissue for three seconds. Repeat the dunking and dabbing procedures for a total of three sets of five dunks followed by a dab.

Then transfer the cover slip into a well containing Tencent formula into fix the cells at room temperature. The color slip is ready for enumeration for long-term storage or to be subjected to counter staining for antibodies or senescence associated beta galacto sase activity as described in the text protocol to enumerate the adhesive endothelial cells, mount the cover slips on microscope slides with DPI counterstain to identify individual cells using an inverted microscope with a fourex or 10 x objective. Acquire images of several fields and count the highest number of monocytes that aggregate on un eradiated endothelial cells.

Set twice this number as the threshold of monocytes on an endothelial cell to be defined as a cluster if required. A different criterion can be used for defining a cluster depending on the nature of the experiment. Count the number of clusters and the number of endothelial cells in the field.

Divide the form by the latter to obtain the percentage of adhesive endothelial cells. Score numerous fields to obtain an average and standard deviation of the counts. Endothelial monolayers at seven days post gray radiation prior to incubation with monocytes were bound by monocytes in clusters around individual endothelial cells.

Although this phenomenon is readily observable under phase contrast, microscopy fluorescent microscopy reveals an even clearer image of the monocyte clusters. After performing the described ADI assay, it is possible to proceed to staining the cells for membrane cytoplasmic or nuclear proteins using appropriate antibodies. With standard immunofluorescence methods, take care with washing steps as excessive force can dislodge the monocytes.

Furthermore, enzyme based assays such as senescence associated becyase can be performed. This causes lysosomes of senescent cells to turn blue, as is evident in the endothelial cell that is selectively bound by numerous monocytes which are identified due to their small size and spherical shape. Since each monocyte cluster corresponds to an individual endothelial cell, enumeration of the clusters reveals the actual number of adhesive endothelial cells within the monolayer and hence the percentage of adhesive endothelial cells within the population.

This is the only method to date that allows quantification of endothelial cell adhesiveness if desired Monocytes attached to endothelial monolayers can be dislodged by a trypsin EDTA solution, and the fluorescence measured using a plate reader as is the case in contemporary methods. Once mastered, this technique can be done in four hours and with very little hands on time. It's important, remember that all the washing and dunking steps must be carried out in an identical fashion and with the same number of repetitions between all the samples Following this procedure.

Other methods such as immunofluorescence and senescence associated beta galacto assays can be performed in order to answer ational questions like what are the characteristics of endothelial cells that are adhesive compared to those that are not.