2.1. Cell Culture

1. Swiss mouse 3T3 cells
Random-bred Swiss mouse 3T3 cells provide optimal feeder support of epithelial cells (4). The cells aremaintained by weekly passage at 1:10 to 1:20. Use fresh cells within 6 months after thawing, since thecells start to senesce or undergo spontaneous transformation with prolonged passages.
2. DMEM culture medium for Swiss mouse 3T3 cells and TECs.
Five hundred mL of Dulbecco’s Modified Eagle’s Medium (DMEM) (D5796, SIGMA) is supplemented
with 1.2 g of HEPES, 0.375 g of L-Glutamine (final 0.750 g/L), 25 mg of Gentamicin, 2 µL of
2-mercaptoethanol, and 25 - 50 mL of fetal bovine serum (5.0 - 10% fetal bovine serum).
3. SMEM culture medium for TECs (note 1)
Five hundred mL of SMEM (SMEM, 11380, Gibco) is supplemented with 1.2 g of HEPES, 0.375 g of
L-Glutamine (final 0.750 g/L), 25 mg of Gentamicin, 2 µL of 2-mercaptoethanol, 5 µL of 50
mg/mL-hydrocortisone hemisuccinate stock solution and 37.5 - 50 mL of fetal bovine serum (7.5-10 %).
4. 50 mg/mL-hydrocortisone hemisuccinate stock solution
100 mg of Hydrocortisone 21-hemisuccinate sodium salt (H2270, SIGMA) is dissolved in 2mL of
H2O.
5. Typsin solution (0.25%)
Solutions of trypsin (0.25%) and ethylenediamine tetraacetic acid (EDTA) (1mM) are purchased from
Gibco/BRL.
You may use TrypLETM Exress (12604, Gibco) instead of Typsin solution (0.25%).
6. IFN-g
IFN-g (#315-05, PeproTech EC Ltd) is dissolved at 106 units/mL in 5.0% FCS-DMEM and stored in 50 µL
of aliquots at -20oC. To enhance the expression of MHC class II molecules on TECs, IFN-g is added to the
5.0% FCS-DMEM at 500 units/mL (note 2).
7. Plastic Wares for cultivation of TECs
The use of Falcon Primaria (60 mm dish, #353802 and 6-well multi-well plate, #353846) or gelatincoated wares (60 mm dish, #4010-020, IWAKI) warrants both the growth and the colony formation of TECs
in both the isolating procedure and the cloning procedure. After the cloning, any other plastic wares fortissue culture as well as the above wares can be utilized to culture TECs.
8. Redcently, the follows reagents from Gibco are available.
GlutaMaxTM-1, 35050: Add 1/40 volumes of this solution into 500 ml of SMEM.
1M HEPES, 15630: Add 1/100 volumes of this solution into 500 mL of SMEM
50mg/mL of Gentamicin, 15750-060: Add 1/1000 volumes of this solution into 500 mL of SMEM.
2.2. Immunofluorescence with Confocal Microscope

1. Confocal microscope

A Zeiss LSM 510 confocal microscope is equipped with a x64/1.4 NA Plan-Apochromat oil-immersion lens.
Ar-laser liner at 488 nm is used for excitation of FITC or Alexa 488. On the other hand, He-Ne laser liner at
545 nm is used for excitation of Rhodamin or Alexa 543. Emission wavelengths are separated by band
pass (505-530 nm) and long pass (585 nm) filters, respectively.
2. Preparation of slide glasses.
1) Eight-hole heavy Teflon-coated slides (Bokusui Brown, New York, NY) are washed extensively with aneutral detergent , rinsed well with hot water, and rinsed once with acetone.
2) Slides are sterilized by rinse with 70% ethanol and dried in a clean bench.
3) Put one drop of 10%FCS-DMEM on each hole of the slides and incubate them in a CO2 incubator forover 24 hr.
3. Phosphate buffered saline (PBS)
Dissolve 9.6 g of the PBS powder (cat. No 05913, Nissui, Japan) in milli-Q grade water to make 1000 mL.
Sterilize PBS by autoclave.
4. Stock PBS (2X)
Dissolve 9.6 g of the PBS powder in milli-Q grade water to make 500 mL. Sterilize stock PBS (2X) byautoclave.
5. Fixation solution
Add 50 mL of milli-Q grade water and 40 µL of 1N NaOH to 4.0 g of paraformaldehyde (P001, TAAB Lab.
Equip. Ltd.). The suspension is swirled and then carefully warmed with a microwave oven at intervals of 10
seconds. These procedures are repeated until the suspension solution turns clear (note 3). Cool thesolution down to room temperature and then add 50 ml of 2X stock PBS. Check that the pH value of thesolution is approximately 7.0, by using a pH-test paper (note 4).
6. Tris-buffered saline (TBS)Dissolve one package of the TBS powder (T903, TAKARA, Japan) with 1000 mL of milli-Q grade water.
7. Quenching solution (0.1M Glycine solution (pH 7.0))
Dissolve 3.75 g of Glycine with 500 mL of milli-Q grade water.

Adjust the pH of the solution to pH 7.0 with

1M Tris-solution.
8. Permeabilization solution (0.05% saponin-TBS)
Dissolve 25 mg of saponin (S-7900, SIGMA) with 50 mL of TBS.
9. Antibody dilution buffer (1% BSA-0.02% NaN3-TBS): Dissolve 1.0 g of BSA powder (Fraction V) and 20
mg of NaN3 with 100 mL of TBS.
10.Mounting medium
Mix one volume of PBS with nine volumes of glycerin (for fluorescence microscopy, cat. no. 1.04095.
MERCK, Germany).
2.3. Antibodies used in the Immunofluorescence Analyses.

1. Anti-pan keratin antibody: A rabbit anti-human keratin antibody (A0575) is purchased from DAKO Co,
CA.
2. Anti-keratin 5 antibody: A rabbit anti-mouse cytokeratin 5 (AF138) antibody is purchased from
CONVANCE, Berkeley, CA.
3. Anti-keratin 8 antibody: A mouse anti-human cytokeratin 8 monoclonal antibody is purchased fromPROGEN Biotechnik GmbH, Heiderberg, Germany.
4. M5/114: M5/114 is a rat monoclonal antibody against mouse I-Ab (5). M5/114 has been purified from theculture supernatant of the hybridoma with the protein A-Sepharose column according to the
manufacturer’s protocol (Amersham Biosciences AB, Uppsala, Sweden). Determine the concentration ofthis antibody by the Bradford method according to the manufacturer’s manual (BIO-RAD Lab., CA).

.

5. In1.1: In1.1 is a rat monoclonal antibody against mouse invariant chains (6). In1.1 is supplied by Dr. G. J.
Hämmerling (German Cancer Research Center, Germany). Add 326 g of ammonium sulfate (55% ofammonium sulfate solution at 0oC ) into 1000 mL of the culture supernatant. After centrifugation at 15,000g
x 15 min, the precipitate is dissolved in a small volume of PBS and dialyzed against 100 times volume of
PBS containing 0.02%NaN3 three times at intervals of more than 4 hours. Determine the antibody
concentration by the Bradford method.
6. 30-2: 30-2 is a mouse monoclonal antibody that reacts with the I-Ab molecules associated with CLIP (7).
30-2 is supplied by Dr. A. Rudensky (Howard Hughes Medical Institute, Univ. of Washington School ofMed., Seattle). 30-2 has been purified from the culture supernatant of the hybridoma with a protein
A-Sepharose column. Determine the antibody concentration by the Bradford method.
7. Anti-H2-DM antibody: Rabbit antisera to H2-DMb2 chain has been raised against a KLH-coupledsynthetic peptide derived from the cytoplasmic tail of H2-DMb2 chain (CRKSHSSSYTPLPGSTYPEGRH)(8). As shown in the sequence of the synthetic peptide, the cystein residue is added to the N-terminal end.
This cystein is available for the conjugation of the synthetic peptide with both KLH for immunization andwith epoxy-activated Sepharose 6B for affinity purification of the antisera. The antisera were purified withthe above synthetic peptide-conjugated epoxy-activated Sepharose 6B, which is made according to the
manufacturer’s protocol (Amersham Biosciences AB, Uppsala, Sweden). Determine the antibody
concentration by the Bradford method. The antibody is designated as anti-H2-DM antibody byimmunofluorescent staining and western blotting.
8. Anti-rab5 antibody: A mouse monoclonal antibody to rab 5 for immunofluorescent study is purchasedfrom Transduction Lab. (Lexington, KY).
9. Anti-rab5 antibody: A rabbit antibody to rab 5A for western blot analysis is purchased from Santa Cruz
Biotech.Inc (Santa Cruz, California).
10. Anti-cathepsin B antibody, anti-cathepsin D antibody, and anti-cathepsin L antibody: These antibodies
are provided by Dr. E. Kominami (Department of Biochemistry, School of Medicine, Juntendo University,
Tokyo, Japan).

11. Anti-cathepsin S antibody: A goat antibody raised against a peptide corresponding to an amino acidsequence at the carboxy terminus of rat cathepsin S is purchased from Santa Cruz Biotech.Inc (cat. No
sc-6505, Santa Cruz, California).
12. anti-LC3 antibody: A rabbit antibody to LC3, a peculiar marker molecule to the autophagy (9) is
provided by Dr. T. Ueno (Department of Biochemistry, School of Medicine, Juntendo University, Tokyo,
Japan).
13. Fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD107a (LAMP-1) monoclonal antibody
is purchased from BD PharMingen.
14. Fluorescent conjugated anti-immunoglobulin (IgGs) antibody: On single-color immunofluorescentstudy, use an anti-IgGs antibody conjugated with Alexa 543 (Molecular Probes, Inc, Eugene, OR) as usual.
On two-color immunofluorescent study, use an anti-rabbit IgG antibody conjugated with TRITC (R 156,
DAKO A/S, Denmark) or Alexa 543 (A11034, Molecular Probes, Inc.) to probe the first antibody and ananti-IgGs antibody conjugated with Alexa 488 (Molecular Probes, Inc,) to probe the secondary antibody.
Upon reaction with the biotin-conjugated second antibody, use avidin-conjugated Alexa 488 (Molecular
Probes, Inc,) to probe it.

3. Methods
3.1 Establishment and Maintenance of TEC Lines
3.1.1. Preparation of Feeder Layer

1. Swiss mouse 3T3 cells is selected to support optimal growth of TECs isolated from mouse thymus. The
cells are cultured with 5% FCS-DMEM at 37oC in 5% of CO2, and maintained by weekly passage at 1:10
dilution.
2. Upon the confluent condition of the cells, remove the medium and rinse the cells once with 0.02%
EDTA-PBS, and then cells are harvested with 0.25% trypsin-0.02% EDTA-PBS. After centrifugation at
500g for 10 min, suspend 1 x 107 cells in 10 ml of 5%FCS-DMEM.
3. To irreversibly inhibit proliferation of the cells, irradiate the cell suspension at 40 Gy.
4. Plate 7 x 105 irradiated cells per 4 ml of 7.5% FCS-DMEM on a 60-mm dish, and culture for 1-2 days.
3.1.2. Establishment of TEC Lines from Mouse Thymus

1. Isolate thymic lobes from embryos at 15 days of gestation or newborn mice and disperse them in PBS
containing 0.25% trypsin and 0.02% EDTA with gentle pipetting.
2. Add an equal volume of 10% FCS-DMEM into the cell suspension. After centrifugation at 500g for 10
min, resuspend the cell pellets in 2 mL of 10% FCS DMEM. Repeat this procedure once.
3. After centrifugation, resuspend the cell pellets at 105 per 1 mL of 10% FCS-DMEM. Plate 1 mL of thecell suspension on Swiss 3T3 cells that are pre-irradiated at 40Gy, plated at 7 x 105 per 2 mL of 10%
DMEM in a 60-mm dish, and culture for 2 days in 5%CO2 at 37oC.

4. Culture the cells in 5% CO2 at 35oC (note 5). Suck out a half volume of the medium and add the same
volume of 10%FCS-Jolik MEM (note 1) at intervals of 4-5 days.
5. The epithelial cells push aside feeder cells and form a colony (Fig. 1). When the colony grows up to
have a diameter of approximately 10 mm, the epithelial cells are cloned by the following procedures.
6. Wash the dish twice with 4 ml of PBS containing 0.02% EDTA to remove Swiss 3T3 cells and thymic
non-epithelial cells. Add 2 ml of PBS containing 0.25% trypsin and 0.02% EDTA into the dish and incubate
the solution to remove the cells from the dish.
7. Add an equal volume of 10%FCS DMEM into the dish, and centrifuge the cell suspension at 500g for 10
min. Resuspend the cell pellet in 2 mL of 10% FCS DMEM. Repeat this procedure once.
8. Suspend the cells at 104 per 1mL of 10% FCS-DMEM in a 60 mm dish. Under an inverted microscope,pick up a single cell or a cluster of cells from the cell suspension by using a micropipette and transfer themto each hole of 24-well plates (Falcon, 3847), which is coated with 105 of 40Gy-irradiated Swiss 3T3 cells
in 1 mL of 10% FCS DMEM. The culture is done in 5%CO2 at 35oC with replacing the cultured medium with
0.5 mL of 10%FCS-Jolik MEM at intervals of 4 days. The cloning procedure is repeated once to obtain
TEC lines.
3.1.3. Maintenance of TEC Lines

1. After establishment of TEC lines, culture TEC lines with 10% FCS-JMEM at 35oC ~ 37oC in 5% CO2.
Replace the cultured medium with the flesh medium at intervals of 4 days. If the growth of the TECs isgetting slow or arrested, add 105 of 40Gy-irradiated Swiss 3T3 cells per 1 mL of 10% FCS DMEM.
2. When the cell growth reaches the confluent condition, cells are washed with PBS(-) once and treatedwith PBS containing 0.25% trypsin and 0.02% EDTA at 37oC.

Add 10% FCS DMEM to inhibit the residual

enzymatic activity of trypsin and then centrifuge at 500g for 10min. Suspend the cell pellet in
10%FCS-JMEM. Replace the one-fourth portion of the cell suspension in a new dish supplemented withthe fresh medium and cultured at 35oC ~ 37oC in 5% CO2
3.1.4. Preservation of TEC Lines

Cells are washed with PBS(-) once, treated with 0.25% trypsin-PBS(-) in 37oC, and centrifuged at 500g
for 10min. Suspend approximately 5 x 106 cells in 1ml of FCS containing 10% DMSO. The stock is first
stored at -70oC, and then transferred into liquid nitrogen 2 days later.
3.2. Immunofluorescence Staining with Antibodies against Keratins and Epithelial Cell Markers for
Characterization of TEC Lines

1. Put one drop (approximately 50 µL) of 105 TEC line cells in 5% FCS-DMEM on eight-hole heavy
Teflon-coated slide prepared as described in chapter 2.2.2, and incubate the slides at 35oC in 5%CO2 forseveral days.
2. To enhance the expression of molecules, including class II MHC molecules, involved in the class II
MHC-restricted antigen presentation, TEC line cells are cultured in 5% FCS-DMEM containing 400 IU/mL

of IFN-g at 37oC in 5%CO2 for 72 hr.
3. Wash the cells twice with FCS-free MEM for 10 min at room temperature
4. Fix the cells with cold acetone (kept at 4oC) for 10 min on ice.
5. Wash the cells three times with cold TBS for 10 min.
6. Incubate the cells with 5 µg/mL of the primary antibody in 1%BSA-TBS overnight in a refrigerator withgentle swinging.
7. Wash the cells three times with cold TBS for 10 min.
8. Incubate the cells with 5 µg/mL of the secondary antibody in 1%BSA-TBS for 3 hr at room temperature.
9. After washing the cells three times with cold TBS for 10 min, seal the slides with PBS (-)-glycerin
solution (1: 9). The sealed slides can be stored at -20oC for at least one month.
8. The immunofluorescence photographs are taken on a Zeiss confocal laser scanning
inverted-microscope, LSM 510.
3.3. Immunofluorescence staining for Localization and Characterization of H2-DM-positive compartments
in TEC Lines

1. Put one drop of 105 TEC line cells in 10% FCS-DMEM on eight-hole heavy Teflon-coated slides
prepared as described in chapter 2.2.2, and incubate the slides at 35oC in 5%CO2 for several days.
2. To enhance the expression of molecules involved in the class II MHC-restricted antigen presentation
including class II MHC molecules, TEC line cells are cultured in 5% FCS-DMEM containing 400 IU/mL of
IFN-g at 37oC in 5%CO2 for 72 hr.
3. A half portion of the cells is further cultured in 5% FCS-DMEM containing 100 IU/mL of IFN-g, 6 µg/ml of
pepstatinA, and 6 µg/mL of E64d at 37oC for 8hr (note 6). Another half portion of cells is further cultured in
5% FCS-DMEM containing 100 IU/mL of IFN-g, without protease inhibitors.
4. Wash the cells once with FCS-free MEM for 10 min at room temperature.
5. Fix the cells with PBS (pH7.0) containing 4% of paraformaldehyde (PFA) for 10 min at room
temperature, and then incubate them in 0.1 M glycine solution (adjusted to pH 7.0 with 1M Tris) to stop thereaction of PFA. .
6. After washing the cells twice with TBS for 10 min, permeabilize the cells with TBS containing 0.05%w/v
of saponin for 10 min at room temperature.
7. Wash the cells three times with TBS for 10 min, apply 5 µg/mL of rabbit anti-H2-DM antibody or anti-LC3
antibody in 1%BSA-TBS onto the cells, and incubate the cells with the antibody overnight in a refrigerator
with gentle swinging.
8. Wash the cells three times with TBS for 10 min, incubate the cells with 5ug/ml of Alexa 543-conjugatedanti-rabbit immunoglobulins in 1%BSA-TBS for 3 hr at room temperature with gentle swinging.
9. Wash the cells three times with TBS for 10 min, incubate the cells with 5 µg/mL of the secondary
antibody in 1%BSA-TBS-0.02%NaN3 overnight in a refrigerator with gentle swinging.

10. Wash the cells three times with TBS for 10 min, incubate the cells with 5 µg/mL of
Alexa488-conjugated anti-IgGs antibody or Alexa488-conjugated avidin in 1%BSA-TBS-0.02%NaN3 for 3
hr at room temperature with gentle swinging.
11. Wash the cells three times with TBS for 10 min, the stained samples were sealed with
PBS/glycerin (1:9) and imaged on a Zeiss LSM 510 confocal microscope.
Notes

1. This medium (JMEM) is a Ca2+-free MEM. The low concentration of Ca2+ is recommended forestablishment and maintenance of TEC lines.
2. The addition of IFN-g to the 5%FCS-DMEM is necessary for the expression of MHC class II molecules
on TEC lines. To fully express the MHC class II molecules, TEC lines must be cultured for 2 days in 5%
FCS-DMEM with 500IU/mL of IFN-g.
3. Paraformaldehyde (PFA) is toxic. Wear gloves, face mask, and safety glasses. Do not boil the PFA
solution with a microwave oven. If so, you must immediately bring the solution into a chemical fume hood.
4. It is better to fix cells with the PFA solution indicating the value of pH 6.8-7.2. Upon fixation with theacidic PFA solution (below pH 6.5), antibodies could not react to the fixed cells efficiently. On the other
hand, upon fixation with the basic PFA solution (over pH 7.5), the extent of background in
immunofluorescent staining slides could increase.
5. The irradiated Swiss mouse 3T3 cells secret growth factors to support the growth of TECs andoppose the adherence of fibroblast cells derived from thymus. The culture of cells at 35oC does
not affect the growth rate of TECs but diminish the growth rate of the fibroblast cells.
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