Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The overall goal of this procedure is to determine the extent of microvascular endothelial cell inflammatory activation by quantitating the relative number of neutrophils that adhere to the endothelial cells in vitro under static conditions. This is accomplished by first treating healthy confluent monolayers of microvascular endothelial cells with the desired experimental stimulus during the second step. Neutrophils from healthy human volunteers are isolated and then labeled with calcium am.

Next, the labeled neutrophils are added to the endothelial cells and allowed to adhere for 20 minutes during which time a pre-wash measurement is obtained by fluorescence spectro geometry. In the final step, the non-adherent neutrophils are washed away, after which a post wash measurement is obtained. Ultimately, the percent and relative adherence of the calcium labeled neutrophils to the endothelial cell monolayers under each treatment condition can be determined.

This method can help answer key questions in the field of human inflammatory biology, since it can determine the relative extent of microvascular endothelial inflammatory activation under different conditions. In addition, this assay has the potential to be used as a tool to study human endothelial based disease processes that involve leukocyte binding to the microvascular endothelium On the day of the assay. Use phase contrast microscopy to confirm that the cells exhibit a healthy cobblestone appearance with little cell debris and that they are 100%confluent.

Once the HM VEC monolayers are ready for treatment, wash the cells once with HBSS and then add 0.3 milliliters of fresh microvascular endothelial cell growth medium with or without an inflammatory agonist to the appropriate wells. Here the HM VEC lung are being treated with TNF alpha for three hours to isolate neutrophils Using Polymorph prep first, bring the density gradient solution to room temperature. Then after collecting 30 milliliters of whole blood from a healthy human volunteer in the presence of an anticoagulant layer five milliliters of the blood over five milliliters of the Polymorph prep in a 15 milliliter conical tube.

Separate the whole blood for 30 minutes at 450 times G and 18 to 22 degrees Celsius with the break off, making sure the tubes are well balanced. Then aspirate the yellow plasma and PBMC containing layers to prevent contamination of the opaque pink neutrophil layer. To remove the neutrophils, aspirate the neutrophil layer with a one to two milliliter serological pipette, while slowly rotating the pipette and tube.

Pull the neutrophils from multiple tubes into a 50 milliliter conical tube containing 30 milliliters of calcium and magnesium free PBS. Bring the volume up to 50 milliliters with more calcium and magnesium free PBS. After spinning down the cells, aspirate the snat and then resus.

Suspend the neutrophils in eight milliliters of sterile water to lice red blood cells after 30 seconds. Quickly add the eight milliliters of cell suspension to two milliliters of five x calcium and magnesium free PBS followed by gentle mixing. After spinning down the cells, again, wash the cells once in calcium and magnesium free PBS then resuspend the pellet in 10 milliliters of RPMI 1640 without phenol red and count the live cells by trian blue exclusion.

After counting, transfer the resuspended cells to a 50 milliliter conical tube and dilute the neutrophils with two to 4 million cells per milliliter with phenol red free RPMI 1640. Then add freshly prepared calcium am working solution to a three micromolar concentration to the cells. Mix the cell suspension by gently inverting the tube several times and then incubate the tube covered in foil at 37 degrees Celsius.

After 30 minutes, centrifuge the labeled neutrophils and then wash the pellet two times in 10 milliliters of 37 degrees Celsius. Phenol red free RPMI 1640, passing the cell suspension through a 40 micron sterile filter to remove any clumps before the second centrifugation. Resuspend the neutrophils in 10 milliliters of 37 degrees Celsius.

Phenol red free RP MI 1640. And then after counting the cells once more, dilute them to 2 million cells per milliliter. Now wash the HM vec monolayers three times with 0.5 milliliters of 0.22 micron filter, sterilized phenol red free RPMI 1640 containing 3%BSA per well after the third wash, aspirate the media and add 600, 000 calcium labeled neutrophils in 0.3 milliliters of cell suspension, or 0.3 milliliters of phenol red free RPMI 1640 without neutrophils to the appropriate wells of the 48 well plate next incubate the co cultures for 20 minutes at 37 degrees Celsius with 5%carbon dioxide, and then just before the end of the incubation period, measure the fluorescence intensity of each well in a spectrophotometer at an excitation wavelength of 485 nanometers and an emission wavelength of 520 nanometers.

Then invert the plate to remove the media and gently padded onto paper towels to remove any remaining liquid. Wash the wells containing the hm ve monolayers and neutrophils five times with 0.5 millimeters per per well of PBS with calcium and magnesium. Removing the PBS in the same way each time.

Refresh the wells with 0.3 milliliters of phenol red free RPMI 1640 per well, and then measure the fluorescence intensity of each well as just demonstrated. Finally, using these formulas determine the percent adherence and relative adherence per well in order to obtain reliable reproducible results using a neutrophil binding assay, it's essential that the health and co fluency of the microvascular endothelial cells are optimal on the day of the assay. Note, the cobblestone appearance and the total co fluency of the monolayers on a Fluor star Optima spectrophotometer.

For example, note that the fluorescence OD 520 nanometers is within the linear range when 10, 000 to 1 million labeled neutrophils are analyzed. Interestingly, as more neutrophils are added to HM VEC lung monolayers treated with an inflammatory agonist, the percent adherence increases. P 38 map kinase has previously been demonstrated to be necessary for e selectin expression.

In TNF alpha treated human endothelial cells and its inhibition reduces the number of adherent neutrophils to demonstrate that the number of neutrophils added to the wells is arbitrary as long as the number of cells utilized is within the linear range of the spectrophotometer. Here a neutrophil adhesion assay with TN F alpha treated HM VEC lung cells in the absence or presence of the P 38 map kinase inhibitor B IRB 7 96 is shown. These results show that the relative decrease in neutrophil binding after P 38 mapk inhibition is similar.

Irrespective of the number of neutrophils per well lectin is expressed on the endothelial cell surface after treatment with TNF alpha and captures neutrophils from the vasculature via electrostatic interactions with glyco molecules present on the neutrophil surface. As shown in this graph, pre incubation of the TNF alpha activated endothelial cells with an antibody against E selectin results in about a 75%reduction in their ability to bind neutrophils. Demonstrating that this assay primarily assesses the very initial events of the neutrophil adhesion cascade in these images, calcium labeled neutrophils bound to untreated and TN ffat treated HM VEC lung monolayers pre incubated with either control IgG or E selectin.

Polyclonal antibody are shown translucent light microscopy images are shown in this column while fluorescence images of the same field of reference using a fluorescein filter set are here in these wells. Representative images of the total neutrophil fluorescence prior to washing with PBS as just demonstrated is shown in these wells. Representative images of the adherent neutrophil fluorescence after washing with PBS as just demonstrated is shown.

Note, the increase in adherence after TN FFA treatment that is reduced in the presence of s selectin polyclonal antibody. While attempting this procedure, it is important to use healthy low passage number endothelial cells and to use the whole blood and isolated neutrophils within two hours of collection.