This method can help answering key questions of basic immunobiology and inflammatory bowel disease research regarding the adhesion cascade of immune cells required for homing to peripheral organs. The main advantage of this technique is that it is a convenient and straightforward approach for answering functional questions in the context of cell adhesion under flow conditions. Although this method was developed for exploring the mechanism of cell poietic anti-integrin antibodies in inflammatory bowel diseases, it can be modified to analyze similar issues in other inflammatory disease settings.
Begin by stretching a piece of rubber tubing on one side with small scissors to allow the insertion of a rectangular capillary approximately 0.5 centimeters into the tube. Then seal the connection with paraffin film. Coat the capillary with 20 microliters of the addressin of interest and seal the side not connected to the tubing with plastic paraffin film for at least one hour incubation at 37 degrees Celsius.
At the end of the incubation, remove the plastic paraffin film and let the liquid soak out of the capillary onto a paper towel to empty the tubing. Then block capillary with 20 microliters of blocking solution for at least one hour at 37 degrees Celsius. It is very important to apply the blocking to the capillary directly after removing the coating to avoid drying up of the capillary as this may impair the functionality of the coded proteins.
While the capillary is being blocked, add 18 milliliters of anticoagulated whole human blood from a 50 milliliter tube and dilute the blood to a total volume of 35 milliliters with PBS. Layer 10 milliliters of density gradient medium under the blood solution and isolate the peripheral blood mononuclear blood cells by density gradient centrifugation. At the end of the separation, transfer the peripheral blood mononuclear cells from the interface of the density gradient medium to a new 50 milliliter tube.
And bring the total volume in the tube up to 50 milliliters with fresh PBS. After centrifugation, resuspend the pellet in 10 milliliters of fresh PBS for counting and stain the cells with a suitable cell tracking dye according to the manufacturer's instructions. At the end of the staining incubation, collect the cells by centrifugation and resuspend the pellet at a 1.5 times 10 to the six cells per milliliter of complete RPMI medium concentration.
Then seed one milliliter of cells into as many wells of a 48 well plate as there are conditions. And add the appropriate volume of anti-integrin antibody to the appropriate wells for a one hour incubation at 37 degrees Celsius. Next use a P1000 pipette to resuspend the cells several times before transferring the cells into individual two milliliter tubes.
Rinse each well with one milliliter of PBS and pool the washes in the appropriate two milliliter tubes. After counting, pellet the cells by centrifugation and resuspened the cells in the appropriate volume of adhesion buffer to obtain 1.5 times ten to the sixth cells per milliliter suspensions. Then add the appropriate volume of manganese chloride to each tube to a one millimolar final concentration.
When the cells are ready, turn on the microscope and select the appropriate filters and lasers, objective and acquisition mode. Remove the plastic paraffin film from the tip of the capillary and use scissors to stretch a five centimeter piece of rubber tubing to allow the capillary to be connected to the smaller tube. Seal the connection between the capillary and the tube with plastic paraffin film.
And mount the capillary on a fixation tray. Place the tray on the tray holder of the microscope so the capillary is in focus. And insert the rubber tubing into the corresponding notch of the peristaltic pump.
Place the unattached end of the tubing into the first sample tube containing the cell suspension. And insert the short tube on the other side of the capillary into a 15 milliliter tube to collect flow through. Let the tubing fill at a 500 microliters per minute flow rate until the cell suspension almost reaches the capillary.
Then let the capillary fill at a flow rate of 100 microliters per minute. When the capillary is filled with cells, adjust flow rate to 10 microliters per minute and capture time lapse images of the cells moving slowly through the capillary along the inside of the addressin coated wall every two seconds for a total of three minutes. Once the cell suspension reaches the inside of the capillary, it is critical to avoid any interruption of the flow as this will lead to static integrin addressin interaction potentially biasing the measurement.
Before discarding stream the whole capillary through the eyepiece of the microscope to make sure that the section observed on the screen is representative. Free the tubing from the pump and let the remaining fluid run back into the two milliliter tube. Then disconnect the capillary and the tubing from the fixation tray and image the next capillary as just demonstrated.
To evaluate the time lapse recordings, open the first sequence in the appropriate analysis software and export the first and last three images as TIFF files. Open the three beginning images in ImageJ and select image, type, and 32-bit to convert the images to 32-bit. Then select image, color and merge channels to merge the images into one image and convert the composite image to RGB for saving as a TIFF file.
After converting the last three images in the same way, count the white cells visible in the beginning and ending composite images. And subtract the number of white cells in the beginning image from the number of white cells in the ending image. The perfusion of capillaries coated with ICAM-1, VCAM-1, MAdCAM-1, but not Isotype Fc chimera with human PBMCs leads to marked adhesion when one of the three addressins is present.
In contrast, the inhibition of integrin addressin interactions using neutralizing antibodies typically leads to a marked decrease of dynamic adhesion that is absent when isotype control antibodies are added. Further, treatment of CD4 human T cells with specific chemokines leads to an increased adhesion to addressins compared to untreated human cells. While attempting this procedure, it is very important to remember that various factors contribute to dynamic cell adhesion in vivo that cannot completely be recapitulated in this assay.
However, it is possible to adjust the assay to address very complex questions. In order to address additional questions on the differential adhesion behavior of testing cell types, it is also possible to perform competitive adhesion assays with different cell types labeled with different cell tracking dyes. After its development, this technique enabled us to explore the effects of the different anti-integrin antibodies used or currently in development for the treatment of inflammatory bowel diseases.
