Dear Spylla Tsiomita,

Protocol

In this study, adult (6 - 8 weeks) female C57Bl/6 mice were used. Mice were purchased from the National Cancer Institute and maintained under specific pathogen free conditions. The University of Minnesota Institutional Animal Care and Use Committee (IACUC) approved all animal experimentation.

1. Preparation of Instruments and Buffers

Prepare enzyme solution (RPMI1640 medium with 0.05% [w/v] of Liberase TH and 100 U/ml of DNase I), albumin-rich buffer (1X PBS [Ca2+/Mg2+-free] with 0.5% Bovine serum albumin and 2 mM Ethylenediamine Tetraacetic acid [EDTA]), FACS buffer (1X PBS [Ca2+/Mg2+-free] containing 1% Fetal bovine serum [FBS] and 0.02% NaN3), FACS sorting buffer (1X PBS [Ca2+/Mg2+-free] containing 2% FBS), and RP10 medium (RPMI-1640 medium supplied with 10% FBS).

Prepare 10 panning plates. Prepare 40 ml coating solution (0.01 mg/ml Goat anti-Rat IgG in 0.1 M NaHCO3 Buffer), and add 4 ml of coating solution into each 100 x 15 mm plate, swirl and incubate at 4 °C O/N. Pour off antibody solution and rinse the plate using 5 ml of 1x PBS for 2 times, swirl vigorously between rinses. Store the prepared plates at 4 °C.

2. Harvest of Thymus Tissue

Euthanize mouse in a CO2 chamber. Put the animal on its back on a dissection mat, pin the feet on the mat and wet the fur by spraying with 70% ethanol.

Make a midline incision through the skin, starting from above the urethral opening straight up to the lower jaw with scissors and extend the incision downward to the knees on both sides. The incision looks like an upside down “Y”. Pull the loose skin back on the sides, and pin it to the dissection mat.

Puncture the diaphragm from the xiphoid, cut the ribs on each side up to about the clavicle, then lift up the ribs with forceps and pin it to the dissection mat. The thymus is just under the ribs, and looks like two thin white lobes overlying the heart (Figure 1). Disconnect connective tissue surrounding the thymus, gently pull and remove the pair of thymus lobes with curved serrated forceps. Place thymus lobes into a 6 well plate containing 5 ml RPMI-1640.

3. Preparation of Thymic Stromal Cells

Trim any surrounding fat and connective tissue and transfer the thymus lobes to a new well of the 6 well plate containing 5 ml freshly prepare enzyme solution.  Make small incisions in the thymus lobes with fine scissors, and incubate the plate at 37 °C for 20 min.

Gently pipette digesting thymus tissue up and down 2 - 3 times using 5 ml pipette for tissue dissociation. Collect supernatant fraction in a 50 ml tube containing 10 ml cold albumin-rich buffer to neutralize enzymes.  Keep the collection tube on ice.  Add 2.5 ml enzyme solution to the remaining tissue and incubate the plate at 37 °C for 15 min.

Perform gentle mechanical agitation with a 3 ml syringe and 18 G needle to break up aggregates.  Pool supernatant into the collection tube.  Add 2.5 ml enzyme solution to the remaining tissue and incubate the plate at 37 °C for 15 min.

Repeat gentle mechanical agitation with a 3 ml syringe and a 25 G needle, and incubate the plate additional 5 - 10 min.

After complete digestion, pool the supernatant into the collection tube.  Centrifuge the collection tube at 400 x g, 4 °C for 8 min to collect the cells.  Aspirate the liquid and suspend the pelleted cells in 10 ml albumin-rich buffer and filter the sample through 100 mm mesh.   Count cells using a hemocytometer.  Then perform flow cytometic analysis (Section 4) or TECs enrichment (Section 5).

4. Identification of TECs by Flow Cytometry

Prepare cells to a final concentration of 4 x 106 cells per 100 µl in FACS buffer for staining in a 96 well round-bottom test plate and keep the plate on ice. Add 20 µl Fc block solution (anti-CD16/CD32 antibody at 10 µg/ml in FACS buffer) to the cells and incubate the cells for 10 min on ice. Spin the plate at 400 x g, 4 °C for 3 min. Discard liquid from wells by flicking the plate face down into a sink.

Suspend the pelleted cells in 100 µl antibody cocktail (anti-CD45, -EpCAM, -MHC class II, and -Ly51 antibodies, and the UEA-1 lectin labeled with different fluorochromes at manufacturer recommended or tested optimal concentrations of each antibody in FACS buffer), and incubate the cells on ice for 20 min in the dark. For compensation, stain 1 x 106 cells with each individual fluorochrome.

Add 100 µl of FACS buffer to each well and spin the plate at 400 x g, 4 °C for 3 min. Repeat the wash step one time, and then resuspend cells in 100 µl FACS buffer. Transfer the cells to a 12 x 15 mm round-bottom FACS tube filled with 300 µl FACS buffer. Acquire samples on flow cytometer and analyze data using FACS analysis software. TEC gating strategy is shown in Figure 2.

5. Enrichment of TECs by Panning

Spin down the thymus cells (prepared in Section 3) at 400 x g, 4 °C for 5 min. Aspirate the liquid and suspend the pelleted cells in FACS sorting buffer at a concentration of 2 x 108 cells per ml in a 15 ml or 50 ml conical tube. Add anti-CD90.2 antibody (Clone 30-H12) (or anti-CD45) at a final concentration of 2.5 µg/ml of cell suspension and incubate cells gently on a rotating mixer at 4 °C for 30 min. Following the incubation, wash with a 5 - 10x volume of RP10 medium and centrifuge the tube at 400 x g, 4 °C for 5 min.

Aspirate the liquid and resuspend the pelleted cells in RP10 medium at a concentration of 1 x 107 per ml. Add 5 ml cell suspension to each coated panning plate (prepared in Step 1.3), swirl and incubate for 30 min at RT. Swirl vigorously; transfer the supernatants into a conical tube. Rinse the plate two times using RP10 medium and pool the supernatants into the collection tube.

Centrifuge the collection tube at 400 x g, 4 °C for 5 min. Aspirate the liquid and resuspend the pelleted cells in 5 ml RP10 medium. Add the cell suspension to a new panning plate, swirl and incubate for 30 min at RT. Collect the cells rinse the plate and pool them into a conical tube. Once TECs are enriched, spin down the cells at 400 x g, 4 °C for 5 min and suspend cells in 5 ml FACS sorting buffer.

6. Purification of TECs by Cell Sorting

Count cells using a hemocytometer and prepare cell suspension at a concentration of 4 x 107 cells in FACS sorting buffer. Add antibodies described in Section 4.4 into cell solution and incubate cells gently on a rotating mixer at 4 °C for 30 min. Following staining, wash and suspend cells to a concentration of 10 x 106 cells per ml of FACS sorting buffer.

Sort TEC subsets through a 100 µM nozzle using fluorescence activated cell sorting. Sorted cell are collected in 30% (v/v) FBS in RPMI-1640). Once cell sorting is done, centrifuge cells at 400 x g, 4 °C for 5 min and prepare for downstream analysis.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4374365/

Hoping this will be helpful,

Rafik

Thank you a lot, I got an idea. However, I am working with T cells and not stromal so I will adjust it.

Thanks a lot Dr. Rafik Karaman for your positive answer .. it's really very helpful, we all highly appreciate your help.