The overall goal of this procedure is to quantify cellular adhesion. This is accomplished by first coating a 96 well microplate with adhesion molecules in the second step. Primary human T cells are isolated from the blood and labeled with CFSE.
Next, the T cells are stimulated with treatments to affect their adhesiveness. In the final step, the treated cells are added to the microplate and adhesion is allowed to occur. Ultimately, the amount of fluorescence per well can be measured to determine the percentage of adherent cells per treatment condition.
The main advantage of this technique over existing method like T-cell addition to model and anterium, is that co culture of T-cell and erl cell is not required. This method can help answer key questions in the immunology field, such as how do specific drugs affect lymphocyte adhesion? Though this method can provide insight into line foot biology, it can also be applied to other maie cells such as neutrophils.
Visual demonstration of this method is critical as the steps are difficult to master and require precision and skill To code a microplate with the adhesion molecules begin by washing 24 wells of a 96 well plate with 50 microliters of wash solution, then incubate each well except the uncoated control wells with 50 microliters of coating solution for one hour at 37 degrees Celsius. Next gently aspirate the coating solution without allowing the pipette tip to touch the bottom of the wells. Then after washing the wells with 50 more microliters of wash solution, incubate the microplate with 50 microliters of adhesion solution per well.
Again, at 37 degrees Celsius. After one hour, gently aspirate the adhesion solution and wash the wells once more with 50 microliters of wash solution To isolate the T cells first thoroughly mix 50 microliters of human T-cell enrichment cocktail per each milliliter of whole blood, and then incubate the treated blood for 20 minutes at room temperature. Next, add six milliliters of the treated blood and an equal volume of calcium and magnesium free PBS enriched with 1%D glucose to a 50 milliliter conical tube at room temperature and mix the solution gently.
Then add 15 milliliters of lympho prep to a fresh 50 milliliter conical tube, and carefully layer their diluted blood sample onto the lymphocyte isolation medium. Separate the cell layers for 20 minutes at 400 times G and 20 degrees Celsius with the break off. Then use a fresh paster pipette to remove the upper layer, leaving the lymphocyte layer undisturbed at the interface.
Transfer the lymphocyte layer to a fresh conical tube with a new pester pipette. Add nine milliliters of PBS to the lymphocytes and then evenly distribute the cells throughout the suspension by gently drawing them in and out. Spin down the cells and then resuspend the pellet in media.
Then dispense the cells into a T 75 culture flask. 20 milliliters of culture media to stimulate the cells after the appropriate culture period. First, add serum free media to 2.4 times 10 to the six T cells, and then serum starve the cells in a 37 degrees Celsius incubator.
After two hours, spin down the cells and resuspend the pellet. In one milliliter of PBS, label the cells with a fluorescent marker in the dark for eight minutes at room temperature, and then stop the reaction by adding 10 milliliters of 37 degrees Celsius PBS. Then after spinning down the cells, resuspend the pellet in 1.2 milliliters of prewarm adhesion solution.
Warm the coated microplate to 37 degrees Celsius, then split the cells into eight 150 microliter aliquots in 1.5. Milliliter tubes stimulate one tube with PMA at 10 nanograms per milliliter to serve as the positive control. Leave three tubes untreated to service the control for stimulation, the unwashed loading control and the control for ICAM one coating and stimulate four tubes with different concentrations of anti CD three antibodies.
Then immediately aliquot 50 microliters of one times 10 to the fifth cells per well of the stimulated cell mixtures into the appropriate wells in the microplate and incubate the plate at 37 degrees Celsius for 15 minutes. To determine the percent of a adherent cells first, wash each well with 150 microliters of warm adhesion solution. Shake the plate gently for a few seconds and then carefully aspirate the medium from each well without touching the bottom of the wells with the pipette tips.
Repeat the wash three times, changing the angle of the pipette with each aspiration. Then turn on the plate reader and open the plate reading software under the task menu. Click new to set up a new protocol.
Then under the actions menu, select the read option. Click on fluorescent intensity and click okay. Enter the excitation wavelength as 485 and the emission wavelength as 528.
Then under the optic option menu, select bottom and click Okay. Then go back to the actions menu and set the temperature to 37 degrees Celsius and click okay. Finally load the microplate and click run.
After exporting the results into a spreadsheet, use the formula to calculate the percentage of adherence cells in these images. An example of an adhesion assay using primary T cells, stimulated with various concentrations of anti CD three antibodies is shown. The unstimulated cells serve as the negative control.
The PMA treated cells serve as the positive control For the anti CD three antibodies, the table displays the relative fluorescence intensities of CFSE. Labeled primary T cells stimulated with different doses of soluble anti CD three antibodies and plated on IAM one coated wells. For every condition, the average intensity of the triplicate wells can be calculated and converted into the relative percentage out of the total cells loaded.
In a typical experiment, the percentage of adherent PMA treated cells is between 40 to 50%while the percentage of unstimulated cells is between five to 10%Note that the percentage of the adherence cells increases as the concentration of anti CD three antibodies used for stimulation increases. Once master, this technique can be performed in under less than three hours if performed properly. While attempting this procedure, it's important to remember to measure the fluorescence of an unwashed well to determine the fluorescence input.
A critical value in data analysis. After watching this video, you should have a good understanding of how to quantify static cellular adhesion. Don't forget that working with human blood samples can be extremely hazardous, and precautions such as wearing a lab coat and gloves should always be taken while performing this procedure.
