Dl dithiotreitol

Spleens and lymph nodes were dissociated using frosted glass slides, while single-cell suspensions of the thymus were prepared using a glass tissue homogenizer. Red blood cells in spleens and thymus were lysed before analysis with Ack lysis buffer (0.15 M NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA). To isolate IEL from the small intestine, the connective tissue, fat, Peyer’s patches and feces were removed. One cm intestine pieces were incubated in RPMI media supplemented with 3% FBS, 5 mM EDTA and 0.145 mg ml⁻¹ DL-Dithiotreitol (Sigma-Aldrich) for 20 min at 37 °C with agitation. The IEL were then isolated by shaking the small intestine pieces in RPMI media with 2 mM EDTA followed by a 30% Percoll (GE Healthcare) gradient of the released cells. For isolation of the thymic epithelial cells, the thymus was first cut in small pieces and pipetted up and down with a wide bore 1000 μl tip until cell release was no longer detectable. The remaining tissue was digested in RPMI media supplemented with 10% FBS, 10 mM HEPES, 0.25 mg ml⁻¹ papain, 0.25 mg ml⁻¹ collagenase D, 0.1 mg ml⁻¹ DNase for 15 min at 37 °C. The TEC were then enriched using EpCAM magnetic microbeads (Miltenyi Biotec) and sorted using a BD FACS Aria III with an 85 μM nozzel.

All reagents used were HPLC grade or electrophoresis grade. Albumin, from bovine serum (BSA), urea, thiourea, 3-[(3-Cholamidopropyl) dimethylammonio]-1- propanesulfonate (CHAPS), β-mercaptoethanol, glycerol 86-88%, bradford reagent, coomassie blue R-250 (CBB), DL-dithiotreitol (DTT), iodoacetamide (IAA), trypsin sequencing trifluoroacetic acid (TFA), sodium deoxycholate (DOC) and acrylamide/bis-acrylamide 30% solution (37.5:1) were purchased from Sigma-Aldrich (St. Louis, USA). (N, N, N’, N’–tetramethylethylene-diamine (TMED), ammonium persulphate (APS), glycine were purchased from NZYTech (Lisbon, Portugal). Ampholytes pH 3–10, formic acid, and ammonium bicarbonate were purchased from Fluka (Steinheim, Germany). Hydrochloride acid (HCl), glacial acetic acid, tris-base, trichloroacetic acid (TCA), sodium dodecyl sulfate (SDS), methanol, acetonitrile were purchased from Panreac (Barcelona, Spain). Bromophenol blue was from Riedel-de Haën. Agarose and Mineral oil were purchased from Biorad (Hercules, USA).
Molecular weight marker for gel electrophoresis NZYColour Protein Marker II was purchased from NZYTech (Lisbon, Portugal). α-Cyano-4-hydroxycinnamic acid puriss for MALDI-MS from Fluka was used as MALDI matrix.