To ensure age matching, we bred mice heterozygously and compared

To ensure age matching, we bred mice heterozygously and compared knockout and heterozygous littermates. Mice were used at 8–12 weeks of age unless otherwise stated. Thymic lymphocytes were isolated by removing the thymus and generating a single cell suspension by straining through a 70 μm wire mesh. Skin lymphocytes were

freshly isolated as previously described [23] with minor adjustments. Briefly, mouse ears were removed at the base, rinsed in 70% ethanol, air-dried, and split into dorsal and ventral halves. The ear halves were placed dermal side down on 0.8% trypsin in PBS (Sigma) and incubated for 30–45 min at 37°C. After enzymatic digestion, epidermis and dermis were separated using forceps. Epidermal MLN0128 sheets were transferred into complete IMDM medium, and dermal sheets were transferred into complete IMDM medium containing 2 mg/mL collagenase IV (Worthington). Skin sheets were shaken for 30 min at 37°C and filtered through a 100 μM cell strainer. Cell suspensions were washed twice with complete IMDM medium before enrichment of lymphocytes using a 40%/70% Percoll gradient. Cells were first blocked with FACS buffer (PBS with 0.5% BSA) containing 1μg/mL anti CD16/CD32 (clone 93, eBioscience).

The following antibodies were used for staining: CD3-PerCP Cy5.5 (145–2C11, eBioscience), TCR Vγ3-allophycocyanin (536, Biolegend), and TCRγ/δ-PE (GL3, BD Biosciences). Dead cells were excluded by propidium iodide staining (Sigma). FACS data were acquired on a Fortessa from BD, using the FACS Diva software. Further analysis was performed using FlowJo from Treestar. Statistics were calculated using GraphPad Prism, selleck chemical where the unpaired Student’s t-test was employed. Mouse ears were removed at the base and hairs were removed with Nair cream. Ears were then split into dorsal and ventral halves. The ear halves were placed dermal side down on 0.5M ammonium thiocyanate and incubated for 40 min at 37°C. Epidermis and dermis were separated using forceps. Epidermal sheets were mounted

on microscopic slides and incubated in 4% PFA for 5 min. After washing, cells were blocked for 30 min with Fc block in PBS containing 10% FCS and 0.1% saponin, followed by incubation with anti TCRγ/δ-PE (GL3, BD Biosciences) for 1 h. Slides were mounted by ProLong® Gold Antifade Reagent (Life Technologies). Ribose-5-phosphate isomerase Supported in part by NIH grants R37 AI047822, R01 DK084647, R01 AI072618, and an award from the Department of Veterans Affairs to ECB. KL was supported by fellowships from the German Research Foundation (DFG), the Crohn’s and Colitis Foundation of America, and the ITI Young Investigator Award from Stanford. This work benefitted from data assembled by the Immgen Consortium. The authors declare no commercial or financial conflict of interest. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset.

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