Our research is focused on neutrophil integrin. In this study, we aim to establish a method to find small molecular antagonists of beta two integrin activation. We believe with this method we can find new antagonists that may deepen our understanding in integrin physiology, and also provide translational insight into integrin-based anti-inflammatory therapy.
In the future, we will focus on discovering new anti-inflammatory drugs and dissecting their mechanisms of action. Using a serological pipette, carefully layer four milliliters of heparinized human blood over eight milliliters of density gradient medium in a 15 milliliter centrifuge tube. Centrifuge the blood at 550 G for 30 to 50 minutes at 20 degrees Celsius.
Then use a one milliliter pipette to remove the upper layer containing plasma and mononuclear cells. Collect neutrophils from the lower cloudy band and approximately three to four milliliters below the clear liquid into a 15 milliliter centrifuge tube containing 10 milliliters of PBS. Gently invert the tube two to three times to mix the neutrophil suspension.
Centrifuge the neutrophil suspension at 400 G for 10 minutes at 20 degrees Celsius. Then carefully decant the supernatant from the tube and resuspend the pellet in five milliliters of PBS. Again, centrifuge the suspension at 300 G for 10 minutes at 20 degrees Celsius.
After removing the supernatant, use vacuum suction to eliminate residual supernatant on the tube wall and around the tube mouth. Resuspend the pellet in one milliliter of neutrophil medium. Combine one milliliter of 1.2 micrograms per milliliter antibody solution with 0.2 milliliters of neutrophil medium in a 1.5 milliliter tube.
Add 25 microliters of antibody solution to the negative control wells in a 384 well plate. In a 15 milliliter tube, combine nine milliliters of the antibody solution, 21.6 microliters of the FMLP solution, and 1.7784 milliliters of neutrophil medium. Add 25 microliters of this mixture to the positive control and test wells in a 384 well plate.
Using a multi-channel pipette, transfer five microliters of compound solution from the compound library plate to the 384 well plate. Centrifuge the suspension at 500 G for one minute. Add 20 microliters of neutrophil suspension to each well using a 16 channel pipette.
Incubate the plate on a shaker at 300 RPM for 10 minutes. Then to fix the cells, add three microliters of 16%paraformaldehyde to each well and incubate on ice for 10 minutes. After turning on the flow cytometer, load the 384 well plate containing the compound treated neutrophil suspension onto the sample holder.
Open the software. Initiate a new experiment. Select 384 well and choose the desired fluorophores.
Then click on plate setup, drag to select all wells, and click on sample. Turn on the high throughput switch, and under the rate panel select high. In the volume box, enter 50.
Select samples in odd numbered columns and activate the agitation switch. Finally, name the samples in the right panel. The mean fluorescent intensity values of the tested compounds were above the cutoff line, indicating that none of the compounds were identified as beta two integrin antagonists.
