Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric Analysis

T-cells are immune cells that protect our body from infectious diseases and cancer. Thymus is a organ that produces T-cells and selects developing T-cells according to the body's self components. We are interested in how the thymus produces and selects T-cells that are protective to our body and yet tolerant to the body.

Our lab has contributed to the analysis of thymic epithelial cells, which have a key role in the production and selection of T-cells in the thymus. Single cell level analysis of thymic epithelial cells has helped us better understand the molecular mechanism of T-cell development and selection. Thymic epithelial cells are highly diverse with complex structures, making their isolation challenging.

Current technology for isolating single cell suspensions is limited and needs improvement. Even so, single cell isolation of thymic epithelial cells enhances our understanding of molecular machinery involved in their development and function. We hope our study will contribute to the understanding of immune cell generation and regeneration.

We believe it will serve as a important cornerstone for advancing human health. After harvesting the thymus from the mouse, place it in five milliliters of RPMI 1640 containing 10 millimolar HEPES in a 60 millimeter plastic dish on ice. Under a dissecting microscope, use curved fine-tip tweezers to remove blood, non-thymus, and fat tissues.

Cut the thymus into small pieces with dissecting scissors. Transfer the thymus pieces into a 15 milliliter tube containing three milliliters of one milligram per milliliter Collagenase and Dispase, and one unit per milliliter DNase in RPMI 1640 containing 2%FBS. Incubate the tube in a water bath at 37 degrees Celsius for 20 minutes.

Then mix the suspension using a three milliliter plastic transfer pipette, and continue incubating for approximately 30 minutes or until the tissue fragments are not seen. Add five milliliters of five millimolar EDTA in HBSS containing 1%FBS. Filter the cell suspension through a 60 micron nylon mesh into a new 50 milliliter tube.

Centrifuge the suspension at 350G for five minutes at four degrees Celsius, and resuspend the pellet in 10 milliliters of FACS buffer. Store the cells on ice and determine cell numbers using a cell counter. After harvesting the thymus from a neonatal mouse, use curved fine-tip tweezers to remove blood, non-thymus, and fat tissues.

Transfer the thymus to a 1.5 milliliter tube containing 0.2 milliliters of RPMI 1640 containing 10 millimolar HEPES. Using a 200 microliter pipette, discard the medium, then add 0.5 milliliters of 0.5 milligrams per milliliter of Collagenase and Dispase and one unit per milliliter of Dnase in RPMI 1640 containing 2%FBS. Incubate the tube in a heat block at 37 degrees Celsius.

during incubation, mix gently using a one milliliter pipette. After 10 minutes, add 0.5 milliliters of five millimolar EDTA in HBSS containing 1%FBS. Filter the cell suspension through a 60 micron nylon mesh into a new 15 milliliter conical tube.

Add nine milliliters of RPMI 1640 containing 10 millimolar HEPES to the tube. Centrifuge the cell suspension at 350G for five minutes at four degrees Celsius, and resuspend the pellet in one to three milliliters of FACS buffer. Store the cells on ice and determine cell numbers using a cell counter.

To begin, add 16 times 10 to the power of 6 neonatal or postnatal mouse thymus cells in a five milliliter polystyrene round-bottom tube. Centrifuge the cell suspension at 350G for five minutes at four degrees Celsius and discard the supernatant. Resuspend the cell pellet in 10 microliters of working concentration of anti-FC receptor monoclonal antibody in FACS buffer.

Incubate the tube at four degrees Celsius for five minutes. Add 40 microliters each of anti-CD45 monoclonal antibody, anti-EpCAM monoclonal antibody, anti-Ly-51 monoclonal antibody, and UEA-1 in FACS buffer. Mix the content of a tube and incubate at four degrees Celsius for 45 minutes.

After incubation, add two milliliters of FACS buffer. Centrifuge the cell suspension at 350G for five minutes at four degrees Celsius, and resuspend the pellet in one microliter of Ghost Dye violet 510 solution. Mix the content of a tube and incubate at four degrees Celsius for 30 minutes.

Now add two milliliters of FACS buffer and centrifuge the cell suspension before fixing the cells with one milliliter of 2%paraform aldehyde for 10 minutes at room temperature. Add 3.5 milliliters of PBS to the tube. Centrifuge the cell suspension at 700G for eight minutes at four degrees Celsius and permeabilize the cells with one milliliter of intracellular fixation and permeabilization buffer.

Mix the content of a tube and incubate at four degrees Celsius for 20 minutes. After incubation, centrifuge the cell suspension and add one milliliter of permeabilization buffer. Again, centrifuge the suspension, before adding 100 microliters each of working concentrations of anti-beta5t antibody or anti-CCL21 antibody.

Incubate for 60 minutes at room temperature. Add one milliliter of permeabilization buffer and centrifuge the cell suspension at 700G for eight minutes at four degrees Celsius. Now add 100 microliters of working concentrations of anti-rabbit IgG and incubate for 30 minutes at room temperature.

After adding permeabilization buffer, centrifuge the cell suspension. Resuspend the cell pellet in 200 microliters of FACS buffer and mix well. Filter the cell suspension through 60 micron nylon mesh prior to flow cytometric analysis.

Flow cytometric profiles of Collagenase and Dispase digested thymus cells from zero day old mice and two week old mice demonstrated that over 90%of particulate signals represented viable cells. Fluorescent signals of viable cells were represented by CD45 and EpCAM. During neonatal stages, cTECs were more frequent than mTECs, but postnatally, mTECs dominated due to low cTEC liberation efficiency.

Fixation and permeabilization of cells allow the detection of intracellular molecules within TECs. Beta5t expression was detected in the majority of cTECs, but not in mTECs. CCL21 was detected in a fraction of mTECs but not in cTECs.

In comparison, air was expressed in a subpopulation of mTECs.