The overall goal of this assay is to determine the effects of treatments of interest on cellular adhesion under shear stress conditions. This method can help answer key questions in the immune cell signaling field, such as, what factors mediate integrin activation. The main advantage of this technique is that single adhesion molecule integrin pairs can be studied in an easily replicated flow system.
Though this method can provide insight into LFA1 activation, it can also be applied to the study of other integrins. Generally, individuals new to this method may struggle because the technical setup must be precisely executed. To harvest the CHO-ICAM cells, treat the culture with 0.5%trypsin EDTA for one minute at room temperature with gentle shaking.
When the cells begin to detach, neutralize the trypsin with four milliliters of fresh medium, and count the number of dissociated cells. Dilute the cells to a 0.75 times 10 to the fifth cells per chamber concentration and spin them down in a centrifuge. Next, resuspend the pellet in 30 microliters per flow chamber, in complete CHO-ICAM culture medium, and slowly seed 30 microliter aliquots of cells per reservoir of the flow chamber.
Settle the cells in a 37 degrees Celsius incubator with 5%carbon dioxide for five minutes. Then add 200 microliters of complete culture medium into both reservoirs of each chamber, and return the plate to the incubator for overnight culture. After harvesting the T cells, count and dilute the cell suspension to a two times 10 to the sixth cells per chamber concentration.
Then centrifuge the cells and resuspend the pellet in one milliliter of PBS, supplemented with 1%glucose per two times ten to the sixth T cells. Next, label the T cells in a 1 to 1000 dilution of CFSE protected from light for eight minutes at room temperature. At the end of the incubation, centrifuge the cells and resuspend the pellet in 240 microliters of serum-free RPMI medium per chamber.
Then split the cells into one 1.5 milliliter tube per each chamber, and store the tubes in a 37 degrees Celsius heat block until loading. Before imaging, warm the microscope live imaging chamber to 37 degrees Celsius, and fill a 500 milliliter glass bottle with water. Make two holes in the lid labelled in and out, and run the syringe connecting input hose through the in hole, and the input reservoir connecting hose through the out hole.
Use a 60 milliliter syringe to flush the input hosing with 60 milliliters of water, followed by 60 milliliters of serum-free 37 degrees Celsius medium to remove any air from the system. When the hosing is primed, transfer the entire input setup into the live imaging chamber of the microscope to maintain the system at 37 degrees Celsius. Run the hosing and syringe out of the chamber, and load the syringe into the syringe pump.
Next, make a hole in the lid of a 250 milliliter bottle to serve as the output waste container and run the output tubing through the hole into the bottle. Then loosely secure the output bottle lid and place the entire output setup into the live imaging chamber. Now place the flow chamber slide onto the microscope stage, and use the 20X objective to set the focal plane on the CHO ICAM mono layer.
Then, beginning with the unstimulated control, discard three 70 microliter volumes from the output reservoir of the first chamber, and add three 70 microliter volumes of CFCS labeled T cells to the input reservoir. While the T cells are moving from the inlet to the outlet, image five random fields of CFSE positive T cells in each chamber to obtain the pre flow cell counts. Next, simultaneously attach the input and output hoses to the flow chamber reservoirs, taking care not to introduce air bubbles, and begin the shear stress flow at 0.3 milliliters per minute, while continuing to visuals the CFSC stained T cells.
As soon as movement of the T cells is observed, start a timer for five minutes, terminating the flow at the end of the five minute period. Then, image five fields of CFSE positive cells in each chamber, choosing the fields randomly to obtain the post flow cell counts. For PMA stimulation, stimulate one tube of T cells with PMA to serve as the positive control immediately before loading the T cells into the second flow chamber for imaging, as just demonstrated.
For SDF-1 alpha stimulation, first remove three 70 microliter aliquots of medium from the output reservoir of the third chamber, and then add 70 microliters of SDF-1 alpha into the input reservoir. After five minutes, discard three 70 microliter aliquots from the output reservoir, and add the remaining tube of T cells for imaging, as just demonstrated. In these representative images, a similar number of T cells are observed pre flow under the different stimulation conditions.
Following five minutes of continuous shear stress, the number of adherent T cells increases under the PMA and SDF-1 alpha stimulation conditions, while a few unstimulated T cells remain adhered. Indeed, SDF-1 alpha T cell activation, for example, induces a near two-fold increase in T cell adhesion, compared to the unstimulated control cells. Further, when the percent adhesion of primary human CD3 positive T cells in the presence or absence of SDF-1 alpha is calculated, 15%of the unstimulated T cells adhere under shear stress, compared to 50%of the T cells under SDF-1 alpha activation conditions.
Once mastered, this technique can be reasonably expanded to six experimental chambers in one assay. When attempting this procedure, it's important to remember to work only with the confluential ICAM mono layer. Following this procedure, other methods, such as cell shape analysis can be performed on the captured images to answer additional questions.
After watching this video, you should have a good understanding of how to quantify cell adhesion under shear stress condition. Thank you for watching this video, and good luck with your experiments.
