The overall goals of this procedure are to isolate and characterize an unconventional intraepithelial cell, gamma delta T cell population from a human endocervical brush sample. This method can help answer key questions in understanding the immune events within the endocervical intraepithelial compartment, to aid in the design of effective strategies for preventing infections. The main advantage of this technique is it provides a powerful and efficient tool for studying endocervical intraepithelial gamma delta T lymphocytes, in vitro.
Demonstrating the procedure will be Laura Romero, a senior research associate from my laboratory. Immediately after collection, place the tube containing the cytobrush on ice. Within one hour of collection, vortex the tube containing the cytobrush for four pulses of 10 seconds.
Then, centrifuge the tube, and carefully remove the brush without disturbing the palette. Next, add one milliliter of IMDM medium to the cells, and place the tube on ice. Place the cytobrush on top of a 100 millimeter cell strainer in a 50 milliliter conical tube, and wash the brush with 20 milliliters of fresh IMDM.
Centrifuge the wash. Resuspending the palette in one milliliter of fresh IMDM, and pool with wash with the cells in the original cytobrush collection tube. To analyze the endocervical cells by flow cytometry, aliquot one to three times 10 to the fifth cells in 100 microliters of fax buffer per five milliliter tube, and label the cells with the antibodies and viability dye of interest for 30 minutes at four degrees celsius in the dark.
At the end of the incubation, wash the cells in one milliliter of the kit buffer. After spinning down the cells, completely decant the supernatant. Use beads and the appropriate IgG control antibodies to set the compensation controls.
Then, load the first tube, and set the gate on the total endocervical cell population in a forward by side scatterplot, followed by exclusion of the cell doublets. Exclude the dead cells according to their viability dye expression, gating on the live CD45 positive and CD3 positive cells. The gamma delta one and gamma delta two positive cells are defined as the subpopulations of CD3 positive cells that express gamma delta TCR.
To isolate the gamma delta T cells by magnetic bead isolation, resuspend up to one times 10 to the seventh freshly harvested endocervical cells in 40 microliters of TCR gamma delta MicroBead kit buffer. Following the manufacturer's instructions for the two-step staining protocol, label the cells with anti-TCR gamma delta hapten antibody, followed by anti-hapten FITC conjugated MicroBeads for 15 minutes at four to eight degrees celsius. At the end of the incubation, wash the cells in one milliliter of the kit buffer, and completely decant the supernatant.
Resuspend the cells in 500 microliters of kit buffer, and apply the cell suspension directly onto a magnetic bead column set in the appropriate separator magnet, collecting the flow-through in a new tube. When all of the cells have run through the column, wash the column three times with 500 microliters of fresh buffer, and pool the washes with the collected cells. To collect the gamma delta T cells, transfer the column into a conical tube, and use the column plunger to flush the T cells from the column with one milliliter of fresh buffer.
To isolate the gamma delta T cells by fluorescence activated cell sorting, label the cells with a viability dye, in the antibody panel as just demonstrated. Then, sort the cells according to their viable dye, and CD45, CD3, and gamma delta positivity. A unique population of gamma delta V1 expressing T cells can be readily detected in human endocervical cell samples.
Detailed phenotypic analysis of the gated CD3 positive gamma delta one positive cells reveals that the majority of these cells are CD4 and CD8 negative. Magnetic bead separation of the endocervical cells, as just demonstrated, results in the purification of a nearly 98%pure population of these TCR gamma delta cells. This level of purity can also be achieved via fluorescence activated cell sorting, validating the use of either technique for the isolation of endocervical gamma delta T cells.
Interestingly, the analysis of endocervical intraepithelial cells, gamma delta T cells, harvested from female HIV infected patients and healthy donor controls, demonstrates a slight reduction in the numbers and viability of these important cells in HIV infected individuals. Once mastered, this technique can be completed in two to three hours if it is performed properly. While attempting this procedure, it's important to remember to quickly process the sample, and to keep the cells on ice throughout the entire procedure, to maintain a good cell viability.
Following this procedure, other methods, like single cell sorting combined with a high-sensitivity throughput multiplex PCR, can be performed to answer additional questions about the inflammatory and signaling pathways within the endocervical intraepithelial compartment. After its development, this technique paved the way for the researchers in the field of HIV and infectious disease to explore novel cell markers of mucosal vulnerability. After watching this video, you should have a good understanding of how to prepare intraepithelial lymphocytes from a human endocervical brush sample.
