The overall goal of this procedure is to measure neutrophil firm adhesion using a flow chamber assay that allows for human neutrophil adhesion in the presence of shear stress. This is accomplished by first preparing a substrate of purified adhesion molecules or an endothelial cell surface, such as human umbilical vein, endothelial cells, or ve. The second step is to separate neutrophils from human peripheral blood using a two layer PHI call centrifugation method.
Next, the flow chamber is assembled to allow for the injection of the isolated human neutrophils under shear stress onto the substrate of either adhesion, ligands or ve. The final step is to record neutrophil adhesion events in the flow chamber, followed by careful quantitation of firm adhesion. Ultimately, the flow chamber assay is used to show firm adhesion of neutrophils toward purified adhesion molecules and a HX surface.
This method can help answer key questions in the autoimmune field, such as functional importance of genetic variations in a tissue molecules that are known to be associated with the development of autoimmunity. Genetic studies in patients with systemic lupus erythema ptosis have demonstrated strong association with variants and Abeta two integrin, a protein involved in neutrophil firm adhesion. We developed this assay system to assess the functional consequences of genetic variation in this beta two endocrine called Mac one on firm adhesion To prepare the culture dishes for use in the flow chamber.
Add one milliliter of a fibrin and gelatin solution to each 35 millimeter tissue culture dish and pipette several times to make sure the entire plate surface is coated. Remove the excess fibronectin and gelatin solution and air dry the plates for at least 30 minutes. To optimize the protein matrix formation, harvest human umbilical vein endothelial cells or qve that have been cultured to 80 to 90%confluence using trypsin EDTA seed 500, 000 cells into each coated tissue culture dish.
Add two milliliters of growth medium to each dish and incubate at 37 degrees Celsius. 5%CO2 cells should be visually inspected daily with medium changes every two days. Allow cells to grow to 80 to 90%confluence.
To begin this procedure, use a marker or pen to draw a circle of 0.5 centimeters diameter at the center of a 35 millimeter tissue culture dish plate. 20 microliters of a 20 microgram per milliliter protein, A solution in the marked area. Use the pipette tip to spread the protein, A solution to cover the whole area within the 0.5 centimeter diameter circle.
It is important to not touch or scratch the surface of the dish. Incubate the tissue culture dishes at 37 degrees Celsius for one hour. Next, wash each protein a coated plate three times with one milliliter of PBS.
After removing the PBS from the third wash plate, 50 microliters of 1%BSA in the marked area to block non-specific binding on the plate. Incubate at four degrees Celsius for two hours. After two hours.
Wash the blocked plate three times with one milliliter of PBS. Prepare the FC adhesion receptor ligand chimeric protein solutions for coating and this experiment and ICAM one FC Kyra at 25 micrograms per milliliter and a P selectin FC Kyra at 0.5 micrograms per milliliter will be used. Coat the marked area with 50 microliters of the substrate, incubate the tissue culture dishes overnight at four degrees Celsius.
The dishes should be used within two days and the coated area should not be allowed to dry out. If necessary. Add PBS to maintain the 50 microliters of solution on the plate.
Neutrophils for this study are isolated from the blood of participants who have given written informed consent. After collecting blood by phlebotomy into an anticoagulant blood collection tube or vacutainer, dilute the blood one-to-one with PBS perform all steps in this procedure. At room temperature, prepare a two layer phy call for separating peripheral blood mononuclear cells or p BMCs and neutrophils in 50 milliliters centrifuge tubes.
First, add 15 milliliters of heavy PHI call and then carefully layer 10 milliliters of light fi call on top of the heavy fi call. There should be a sharp border between the light fi call and heavy fi call layers. Finally, carefully layer 25 milliliters of the diluted blood sample on top of the light fi call without disturbing the PHI.
Call layer centrifuge the tubes for 30 minutes at room temperature after centrifugation, multiple layers should be present. The neutrophil layer with few red blood cells is between the light and heavy phi. Call using a transfer pipette harvest and transfer the neutrophil layer into a new 50 milliliter tube and add PBS to a final volume of 50 milliliters.
Centrifuge at 225 times G for 10 minutes. At room temperature after centrifugation, there may still be red blood cells mixed with the neutrophils. Aspirate the supernatant down to the 10 milliliter.
Mark Resus. Suspend the neutrophil red blood cell pellet by briefly, vortexing at low speed and then wash again with 50 milliliters of PBS aspirate the supernatant to remove the contaminating red blood cells. Resuspend the pelleted cells in the residual PBS by a brief low speed vortex.
Add 25 milliliters of water and gently vortex for 10 seconds. To lice. The red blood cells add 25 milliliters of 1.8%sodium chloride and immediately mix by centrifugation at 225 times G for 10 minutes.
The contaminating red blood cells should now be lyed leaving a white neutrophil cell pellet. Wash the neutrophil cell pellet with PBS, discard the supernatant and resuspend the isolated neutrophils in RPMI medium with 10%FBS. After determining the cell concentration under a light microscope with a hemo cytometer, adjust the cell density to 500, 000 cells per milliliter with complete RPMI 10%FBS medium.
Prior to starting the flow chamber adhesion assay prime the VE with 20 nanograms per milliliter of human TNF alpha for four to six hours to upregulate and stimulate adhesion molecule expression. When the VE are ready, assemble the flow chamber. Place the 35 millimeter dish containing the confluent VE on the microscope table.
For the purposes of this video, only neutrophil adhesion to VE will be examined, but the same procedure applies to studying neutrophil adhesion to purified adhesion molecules connect the parallel plate flow chamber with the syringe pump and vacuum system and leave one line open for the neutrophil input. Insert the flow chamber on top of the plate and fasten the flow chamber assembly. Start the video recording program on the computer connected to the microscope.
Adjust the field and focus of the microscope until a clear field with fully grown VE is visible. Using the syringe pump, rinse the flow chamber with RPMI medium. Make sure there are no air bubbles within the chamber or the neutrophil input line.
Use the syringe pump to inject the neutrophils into the flow chamber at defined speeds. Record the video because neutrophil adhesion can occur rapidly, a four to five minute video is usually sufficient to quantitate adhesion events for analysis. This video clip shows an example of neutrophils binding to a HU E coated flow chamber.
An adherent cell is defined as a cell that moves less than one cell diameter within five seconds. On the HU e coated surface. Shown here are screenshots at different time points from sample videos of neutrophils adhering to an ICAM one P selectin coated surface, or a uve coated surface.
In both experiments, the neutrophil flow speed is 350 microliters per minute with a neutrophil density of 500, 000 cells per milliliter. Under typical conditions, it was observed that 50 to 70 human neutrophils firmly adhered to the ligand or VE coated surface during a four minute recording period. However, allelic variants of neutrophil adhesion molecules or allelic variants in the substrate could substantially alter quantitative neutrophil adhesion by recording similar length videos with different donors.
Neutrophils, the number of adherence cells per minute can be calculated to compare the adhesion properties between different donors. While attempting this procedure, it's important to remember to visually check the neutrophils for cell clamping caused by isolation induced cell activation. In addition, parallel flow cytometric assessment of cell activation can be performed to ensure the activation state of cells is matched between donors and between experiments.
This technique paved the way for researchers in the field of autoimmunity to explore cell adhesion in primary human cells from genotype donors that have different coding region alleles of the CD 11 B chain of the beta two integrin, MAC one. These studies have allowed for a careful and quantitative assessment of the functional impact of these allelic variants in the human system.
