The approach that we're gonna be talking today about has been derived to compare properties and functional activities of tissue-derived and peripheral blood T cells. The significance of the topic is exemplified by the fact that peripheral blood T cells represent only 20%of the total T cells in a body. Being able to study a small number of tissue-derived T cells is very essential, and that's exactly what we have achieved.
The main advantage of this technique is that it requires smaller number of cells and smaller volumes of reagents than traditional techniques. Begin by using polypropylene scissor-type forceps to submerge glass coverslips in freshly-prepared acidic piranha solution for 25 to 30 minutes. At the end of the wash, rinse the coverslips seven times in a fresh volume of ultra-pure water for each wash.
Then set the wet coverslips aside to let the remaining water roll off the clean glass. When the coverslips are dry, aliquot two microliters of the final liposome mixture precisely in the center of a self-adhesive slide channel within a sterile flow hood. And immediately but carefully align one clean and dry coverslip with the slide to allow the coverslip to be gently lowered onto the sticky side of the slide.
And use the outer ring of the polypropylene scissor-type forceps to apply a gentle pressure to the peripheral contact of the coverslip with the slide, making sure that the slip is tightly attached to the slide to prevent leakage. Then turn the slide over, and use a permanent marker to draw four dots around the bilayer on the external side of the slide assembly. Before the first injection, designate one port of the channel as the entry port, and the other as the exit port, maintaining this designation throughout the experiment.
To avoid bubble formation, insert the end of the pipet tip directly into the entry port until you feel the rubber seal of the slide channel being penetrated with the tip. Slowly fill the channel with 50 microliters of warm assay buffer. Inject 200 microliters of nickel(II)chloride in Casein blocking solution into the entry port.
And remove 50 microliters of the buffer from the exit port. After a 45-minute incubation, remove the excess Casein solution from the exit port. Inject 100 microliters of an ICAM-1 in streptavidin solution into the entry port for a 45-minute incubation at room temperature.
At the end of the incubation, remove the excess protein solution from the exit port. And wash the bilayer two times with 100 microliters of assay buffer per wash. Then label the bilayers with 100 microliters of fluoro-4-conjugated anti-CD3 antibody for 45 minutes at room temperature.
Followed by two washes in 100 microliters of assay buffer per wash as demonstrated. To image T cell bilayer interactions, place the slide on the 37-degree-Celsius heated stage of a total internal reflection fluorescence, or TIRF microscope. And adjust the stage using the ink marks to center the bilayer onto the 100-power objective.
For granule release imaging, add fluorescence-conjugated anti-CD107A antibody FAB fragments to the CD4 T cell suspension. And resuspend the labeled cells in 50 microliters of assay buffer for injection into the entry port of the slide channel. Then select the appropriate number of experimental fields, and record images of each field once every minute for 30 minutes after the injection.
In this representative experiment, no differences between CD8 T cells that formed classical immunological synapses on the lipid bilayers built in either the flow cell or the multi-channel flow slide were observed. Upon the lipid bilayer interaction, CD4 T cells established mature immune synapses, but either do or do not release granules, release granules without the formation of mature synapses, or neither form synapses nor release granules. Of note, peripheral blood mononuclear cell-derived CD4 T cells are able to begin releasing granules almost twice as quickly as can lymph node-derived CD4 cells.
While attempting this procedure, it is important to maintain constant temperature and pipet slowly so that there are no bubbles formed. In addition to our capabilities to investigate a T cell bilayer interface, this approach would also allow us to perform an intercellular staining of the T cells that touched the bilayer. Which would significantly facilitate an amount of information that can be used to describe particular T cells derived from either lymphoid tissue or other tissues.
Don't forget that working with piranha solution is extremely hazardous, and it is crucial that you wear proper PPE while attempting this procedure.
