The overall goal of this assay is to quantify the capacity for monocytes from human clinical samples to undergo transendothelial migration and foam cell formation. This method can be used to answer key questions in the cardiovascular field such the atherogenic potential of cells from individuals who are at risk of acute coronary artery disease. The major advantage of this technique is that clinical samples can be measured both from fresh whole blood or stored patient cohorts.
Furthermore, foam cell formation can be quantified by both microscopy and flow cytometry. First, prepare the polymerized collagen gels from a fresh collagen suspension. To a five-milliliter polystyrene tube, add sodium hydroxide, then 10 times M199, then acidic acid, and finally, add fibrous collagen.
Mix this well by pipetting. Next, aliquot 50 microliters into the internal wells of a sterile flat-bottomed 96-well tissue culture plate. In the outer edges of wells, load 200 microliters of one times Dulbecco PBS to prevent desiccation.
After two hours of incubation at 37 degrees Celsius, the collagen will polymerize. Then, overlay the gels with 150 microliters of one times supplemented M199 and incubate them until needed for use five days later. To expand stored human umbilical vein endothelial cells, prepare a 25-square-centimeter culture flask with one milliliter of fibronectin solution and incubate the dish for 10 minutes at room temperature.
Meanwhile, thaw the stored cells in a water bath to resuspend them in M20. Once the cells are thawed, aspirate any residual fibronectin solution from the dish and plate the cells. Then, culture to confluence by replacing the media after two to three days.
The HUVECs should be confluent on day five of culturing. Detach them by aspirating the culture's supernatant from the flask, and then wash away the serum-containing media using two to three milliliters of one times PBS. Then, add five milliliters of diluted trypsin and incubate the cells briefly at room temperature shaking the cells gently until they appear detached.
Then, quickly collect them with five-milliliters of M20 and transfer the suspension to a 10-milliliter tube and centrifuged cells. Now, resuspend the cells in M20 at 200, 000 cells per milliliter. Then, aspirate the M199 from the prepared collagen plate and add 100 microliters of HUVECs to each gel.
Continue the culture for three days. On day eight of culturing the HUVECs, activate the monolayers prior to adding the monocytes. After aspirating the overlaying media, add 100 microliters of human TNF-alpha solution to each well.
Then, incubate the plate at 37 degrees Celsius for four hours. After the incubation, remove the media and wash the gels twice using 100 microliters of M199 per wash. Now, add 50, 000 freshly isolated monocytes or purified monocyte subsets in 100 microliters of M20 to the HUVEC monolayers.
After adding the monocytes, incubate the plate for an hour to allow for forward transmigration of the cells. After an hour, collect the non-transmigrated cells in wash solution. Start by pooling two EGTA washes using 100 microliters per well.
Then, spin down the combined wash solutions at 300 G for five minutes at four degrees Celsius. Discard the supernatant, and resuspend the cells in 30 microliters of PBS and count the cells to determine the percentage of forward transmigrated cells. Now, wash plate wells once with M199 and add 100 microliters of fresh M20 to the plate wells and incubate the plate for two days.
After two days, repeat the procedure for collecting the non-transmigrated cells in EGTA wash solution in order to collect reverse transmigrated cells and proceed with phenotypic analysis of the cells. To phenotypically characterize the gel-bound cells by FACS analysis, digest the cells by adding 100 microliters of 37-degrees-Celsius collagenese D and incubating for 20 minutes at 37 degrees Celsius. Following the incubation, macerate the gels using a 200-microliter pipette tip and incubate them for a further 20 minutes to fully digest the gels.
Once fully digested, filter and pool the digested replicate gels through a 35-micron nylon mesh into a FACS tube on ice. Now, wash the filtered cells once with cold FACS wash solution. Centrifuge the cells and resuspend them in 100 microliters of cold wash solution.
Then, perform FACS staining and analysis according to the text protocol. To analyze the cells by microscopy, first stain the cells as described in the text protocol. To detach the stained cells, rim the wells using a 21-gauge needle with the bevel facing outwards.
Next, prepare slides to mount the gels. Punch two small holes into a strip of double-sided tape and then fix the tape to the slide and remove the protective coating. Next, add a drop of water to each hole in the tape.
Then, using tweezers, gently transfer two gels onto the two holes and cover the tape. Finally, carefully attach a coverslip to the tape and proceed with visualization of the cells. Following transmigration, non-transmigrated cells were counted as described.
A total of 15, 000 non-transmigrated cells were recovered, and the percentage of forward transmigration was determined to be 95%Following 48 hours of further incubation, 36, 000 reverse transmigrated cells were recovered which amounted to 12.6%reverse transmigration. Staining the gels with oil red O revealed foam cells pointed out by white arrows and macrophages pointed out with black arrows. These monocytes were treated with oxidized LDL during transmigration, a substance used to induce foam cell formation in conventional foam cell induction models.
Aggregate cell count data from 12 donors confirmed that the incubation of monocytes with oxidized LDL induced more foam cell formation than when monocytes are incubated with native LDL or M199 media alone. The transmigrated cells were also analyzed by flow cytometry. After excluding dead cells, doublets and CD45 negative HUVECs, the major population of migrated cells was selected and the mean fluorescence intensity of the receptors of interest was compared with the fluorescence minus one or isotype control data.
After watching this video, you should have a good understanding of how to use this assay to quantify monocyte transendothelial migration and foam cell formation from human clinical samples. Don't forget that working with human clinical samples can be extremely hazardous and good laboratory technique must always be used.
