Bindloss et al Saline(PBs, Gibco) before measuring A600. An aliquot of the washed cells was permeabilized by vortexing in 0. 1% SDS and chloroform. Nitrate reductase activity was then assayed by adding equal volumes of 2 mM methyl viologen and bicarbonate/dithionate nitrate solution( 8 mg/mL NaHCO3, 8 mg/mL Na2S2O4, 0.5 M NaNO3 ). The reduction of nitrate to nitrite was allowed to progress for 5 minutes before vortexing vigorously to terminate the assay. To visualize the amount of nitrite produced, equal volumes of sulfanilic/HCI(1% Sulfanilic Acid, 20% HCI) and Marshalls Reagent(0. 13%N-1 napthylethylenediamine 2 HCI)were added, vortexed and incubated for 10 minutes at temperature. The A540 and A420 were measured and relative activity units were detern with the following equation units=([A540-(0.72 x A420)(t x V X A600)) x 100 Hy poxia staining For detection of hypoxia, mice were treated with 60mg/kg of pimonidazole HCl ip HypoxyprobeTM-I kit, Hypoxyprobe)one hour prior to euthanasia. Colon samples were fixed in 10% buffered formalin and paraffin-embedded tissue was probed with mouse anti pimonidazole monoclonal IgGl (MAb 4.3.11.3)and then stained with Cy-3 conjugated goat anti-mouse antibody (Jackon Immuno Research Laboratories). Samples were counterstained with DAPI using SlowFace Gold mountant. Samples were scored based on the degree of colonic epithelial hypoxia(0: no hypoxia; 1: mild focal hypoxia; 2: moderate multifocal hypoxia; 3: intense diffuse hypoxia) Representative images were obtained using a Zeiss Axiovert 200 M fluorescent microscope and brightness adjusted( Adobe Photoshop CS2) Flow cytometry Preparation of mouse intestinal lymphocytes has been described previously(35). After initial solation, intestinal lymphocytes preparation was enriched for T cells using a CD3 enrichment kit(EasysepTM mouse T cell enrichment kit, STEMCELL technologies) according to manufacturers instructions. T( CD3T)cell-enriched intestinal cell population was resuspended in 2 ml Dulbecco's phosphate-buffered saline(PBs)without calcium and magnesium and stained with aqua live/dead cell discriminator(Invitrogen) in accordance with the manufacturer's protocol. Cells were then rinsed and stained for Treg population using a Treg specific kit( Mouse regulatory T cell staining kit #2, eBioscience)according to manufacturer's instructions Stained cells were analyzed with an Lsr II flow cytometer (Becton Dickinson, San Jose, CA). The data were analyzed by using FlowJo software (Tree Star, Inc., Ashland, OR). Gates were set on singlets and then on live cells. Subsequent gates were based on fluorescence-minus-one and unstained controls Fig S6) actate measurements For determination of intracellular lactate measurements, primary colonocytes were isolated as described above. Lactate measurement in colonocyte lysates was performed by using a Lactate Colorimetry Assay Kit Il(Biovision, Milpitas, CA) according to manufacturers Instructions Science Author manuscript; available in PMC 2017 October 1Saline (PBS, Gibco) before measuring A600. An aliquot of the washed cells was permeabilized by vortexing in 0.1% SDS and chloroform. Nitrate reductase activity was then assayed by adding equal volumes of 2 mM methyl viologen and bicarbonate/dithionate/ nitrate solution (8 mg/mL NaHCO3, 8 mg/mL Na2S2O4, 0.5 M NaNO3). The reduction of nitrate to nitrite was allowed to progress for 5 minutes before vortexing vigorously to terminate the assay. To visualize the amount of nitrite produced, equal volumes of sulfanilic/HCl (1% Sulfanilic Acid, 20% HCl) and Marshall’s Reagent (0.13% N-1- napthylethylenediamine 2 HCl) were added, vortexed and incubated for 10 minutes at room temperature. The A540 and A420 were measured and relative activity units were determined with the following equation: units = {[A540 – (0.72 × A420)]/(t × v × A600)} × 100. Hypoxia staining For detection of hypoxia, mice were treated with 60mg/kg of pimonidazole HCl i.p. (Hypoxyprobe™-1 kit, Hypoxyprobe) one hour prior to euthanasia. Colon samples were fixed in 10% buffered formalin and paraffin-embedded tissue was probed with mouse anti￾pimonidazole monoclonal IgG1 (MAb 4.3.11.3) and then stained with Cy-3 conjugated goat anti-mouse antibody (Jackon Immuno Research Laboratories). Samples were counterstained with DAPI using SlowFace Gold mountant. Samples were scored based on the degree of colonic epithelial hypoxia (0: no hypoxia; 1: mild focal hypoxia; 2: moderate multifocal hypoxia; 3: intense diffuse hypoxia) Representative images were obtained using a Zeiss Axiovert 200 M fluorescent microscope and brightness adjusted (Adobe Photoshop CS2). Flow cytometry Preparation of mouse intestinal lymphocytes has been described previously(35). After initial isolation, intestinal lymphocytes preparation was enriched for T cells using a CD3 enrichment kit (Easysep™ mouse T cell enrichment kit, STEMCELL technologies) according to manufacturer’s instructions. T (CD3+) cell-enriched intestinal cell population was resuspended in 2 ml Dulbecco’s phosphate-buffered saline (PBS) without calcium and magnesium and stained with aqua live/dead cell discriminator (Invitrogen) in accordance with the manufacturer’s protocol. Cells were then rinsed and stained for Treg population using a Treg specific kit (Mouse regulatory T cell staining kit #2, eBioscience) according to manufacturer’s instructions. Stained cells were analyzed with an LSR II flow cytometer (Becton Dickinson, San Jose, CA). The data were analyzed by using FlowJo software (TreeStar, Inc., Ashland, OR). Gates were set on singlets and then on live cells. Subsequent gates were based on fluorescence-minus-one and unstained controls Fig S6). Lactate measurements For determination of intracellular lactate measurements, primary colonocytes were isolated as described above. Lactate measurement in colonocyte lysates was performed by using a Lactate Colorimetry Assay Kit II (Biovision, Milpitas, CA) according to manufacturer’s instructions. Byndloss et al. Page 13 Science. Author manuscript; available in PMC 2017 October 16. Author Manuscript Author Manuscript Author Manuscript Author Manuscript