Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The overall goal of this procedure is to isolate, identify and purify thymic epithelial cells or tech. This is accomplished by first enzymatically, digesting and mechanically disrupting the thymus to dissociate the tech into a single cell suspension. Next, the tech can be analyzed by flow cytometry or enriched via a panning strategy.

Ultimately, thymic epithelial subsets can be purified by fluorescence activated cell sorting. This method can help answer many key questions in the field of T-cell development, such as, how do thymic epithelial cells promote thymic selection? What causes thymic dysfunction in aging animals and how can we achieve T-cell reconstitution in vitro?

The main advantages of this technique are the high recovery of variable emmic germa cells, the unbiased enrichment of the emmic epithelial cells, and the high purity that can ultimately be achieved by cell sorting. To isolate thymic stromal cells, first identify the thymus, which is just under the ribs and looks like two thin white lobes overlying the heart. Then disconnect the connective tissue surrounding the thymus and use curved serrated forceps to gently pull and remove the thymic lobes.

Place the lobes into a six well plate containing five milliliters of medium and trim any surrounding fat and connective tissue. Then transferred the trimmed lobes to a new well containing five milliliters of freshly prepared enzyme solution and use fine scissors to make small incisions in the tissue. Place the plate in a 37 degree Celsius incubator.

After 20 minutes, gently pipette the tissue up and down several times with a five milliliter pipette to dissociate the slurry into a single cell suspension. Collect the supernatant fraction in a 50 milliliter tube containing 10 milliliters of cold albumin rich buffer on ice to neutralize the enzymes. Then add 2.5 milliliters of enzyme solution to the remaining tissue.

Incubate the plate for 15 minutes. After that, gently agitate the tissue with a three milliliter syringe equipped with an 18 gene needle to break up any aggregates. Then transfer the to the collection tube.

Then add 2.5 milliliters of enzyme solution to the remaining tissue. Incubate the plate for 15 minutes. After the third incubation, repeat the mechanical agitation with a 20 5G needle.

After incubating the tissue for a final five to 10 minutes, transfer the supernatant to the collection tube for centrifugation to identify the recovered tech by flow cytometry Following standard antibody staining procedures, run the samples on a multi-parameter flow cytometer and analyze the data using the appropriate fax analysis software to enrich the tech further by the panning method. First, incubate the cells with the appropriate antibody for the enrichment. Next, add the cell suspension to a pre-coded panning plate and then swirl the plate and incubated at room temperature.

After 30 minutes, swirl the plate vigorously and then transfer the supernatant to a conical tube. Rinse the plate two times with fresh, medium, pooling the washes in the collection tube. Then after spinning down the cells, resuspend the pellet in five milliliters of fresh, medium and transfer the cell suspension to a new panning plate, collecting the cells after swirling and incubating as just demonstrated to further purify the enriched tech into their thymic cell subsets.

After labeling with the appropriate antibodies, sort the tech through a 100 micrometer nozzle by fluorescence activated cell sorting, collecting the cells in 30%volume per volume FBS in medium. In this representative gating strategy for identifying tech by flow cytometry the expression of epca, but not CD 45 by the tech can be observed. Thus, tech can be gated according to their ep, camm and CD 45 expression tech are comprised of cortical or CEC and medullary or mtech thymic epithelial subsets.

These subsets can be distinguished by the live 51 antibody, which recognizes G glutamyl amino peptidase expressed by ctec and the lectin UEA one, which binds to mtech both ctech and Mtech express MHC Class two on their surfaces. Although mtech can be further classified into mtech low and mtech high populations, depending on their level of MHC two expression, approximately eight times more tech can be recovered using the just demonstrated liase enzyme based protocol compared to a protocol with collagenase and disc space with about nine times 10 to the fifth, able to be recovered from just one mouse thymus to decrease the sorting time and to increase the viability of the recovered stromal cells. Panning can be used as just demonstrated to deplete the thymocytes sites compared to whole prepared thymus cells.

The proportion of tech increases from less than 0.5%to over 15%in the post enriched cell population. After panning, the proportions of the tech subsets are not altered, suggesting that neither subset is selectively lost during panning. Compared to the preen enrichment sample, the recovery rate of tech is about 85%After watching this video, you should have a good understanding of how to enrich themic germ cells from more thans by panic, as well as how to identify and purify more themic epithelial subsides by flow cytometry.