Real-time PCR. Total RNA was isolated from purified splenic or LP macrophages using
|
|
- Flora Jefferson
- 6 years ago
- Views:
Transcription
1 Supplementary Methods Real-time PCR. Total RNA was isolated from purified splenic or LP macrophages using the Qiagen RNeasy Mini Kit, according to the manufacturer s protocol with on-column DNase digestion using the RNase-Free DNase set. cdna was generated using the Superscript First-Strand Synthesis System for RT-PCR and random hexamer primers (Invitrogen), according to the manufacturer s protocol. cdna was used as a template for quantitative real-time PCR using SYBR Green Master Mix (BioRad), and gene-specific primers (Supplementary Table 1, online). PCR and analysis was performed using a MyiQ ICycler (BioRad). Gene expression was calculated relative to Gapdh. Stimulation of lymphocytes. In vivo: B6.PL (Thy.1 + ) recipient mice were reconstituted with 2.5 x 10 6 OT-II TCR transgenic CD4 + T cells (Thy1.2 + ) one day prior to receiving 2x10 6 OVA-pulsed splenic or LP CD11b + cells in the footpad. Four days later, DLN cells were counted, stained with antibodies and analyzed by flow cytometry. Data were acquired on a FACSCalibur (BD Biosciences) and analyzed with FlowJo software. In vitro recall responses were assayed by restimulating total LN cells (5x10 6 /ml) with relevant OVA peptides. In vivo depletion of LP DCs. CD11c-DTR mice were fed on day 0, 2, 4, 6, and 8 with 500ng/mouse of diptheria toxin (Sigma) and on day 10 small intestinal LP lymphocytes were isolated and either stained directly or stimulated in vitro for 5 h with PMA (50ng/ml) and ionomycin (500ng/ml) in the presence of GolgiStop (PharMingen) before staining and flow cytometric analysis.
2 Electron microscopy. For scanning electron microscopy analysis, sorted CD11b + CD11c splenic or intestinal cells were fixed with 2.5% (volume/volume) glutaraldehyde in PBS, ph 7.4, were processed and analyzed at the Histology and Electron Microscopy Core Lab at the Yerkes National Primate Research Center of Emory University. Cytokine detection. IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40, IL-12p70, IL-17 and IFN-γ in culture supernatants were detected by a standard two-site sandwich ELISA for cytokines as described in the BD PharMingen protocol. For T cell cultures, supernatants were harvested after 72 h. For intracellular cytokine analysis, cells were stimulated with 10µg/ml plate-bound α CD3 and 1µg/ml α CD28 for 6 h in the presence of GolgiStop (PharMingen) and subsequently incubated with blocking 2.4G2 anti-fcγriii/ii (PharMingen) and stained with PerCP-conjugated anti-cd4. Cells were permeabilized by using Cytofix/Cytoperm and stained by using fluorochrome-conjugated antibodies (PharMingen) according to the manufacturer's protocol. Flow cytometry. Isolated splenocytes or small intestinal LP cells were resuspended in PBS containing 5% FBS. After incubation for 15 min at 4 C with the blocking 2.4G2 anti-fcγriii/i, the cells were stained at 4 C for 30 min with labeled antibodies. Samples were then washed two times in PBS containing 5% FBS. The samples were immediately analyzed at this point, or they were fixed in PBS containing 2% paraformaldehyde and stored at 4 C. Antibodies used for analysis: FITC-labeled anti-mouse CD1d, CD13, CD24, CD25, DEC205, ICAM2, VCAM1, Ly6C; PE-labeled anti-mouse CD19, B220,
3 Gr-1, I-A b, CD40, CD80, CD86, PD-L1, PD-L2, CD14, CD44, CD45RB, CD62L, CD69, CD103, 33D1, Fas, CD135; PerCP-labeled anti-mouse CD3ε, CD4, CD8α, CD45; APClabeled anti-mouse NK1.1, CD11c, TCRß (PharMingen), APC-labeled anti-mouse F4/80 (ebioscience), and PE-labeled anti-mouse CX 3 CR1 (MBL). Intracellular FoxP3 staining was performed using PE-labeled anti-mouse FoxP3 (clone FJK-16s; ebioscience). Flow cytometric analysis was performed on a Becton Dickinson FACSCaliber flow cytometer at the Emory Vaccine Center. Frozen section immunofluorescence analysis. Mice were sacrificed and spleens or small intestines were snap frozen in tissue-freezing medium (Triangle Biomedical Sciences). Immunofluorescence staining was carried out on 6-µm sections. Sections were air dried and fixed with a 1:1 acetone methanol mixture at 20 C for 10 min followed by blocking for 30 min with TNB buffer (PerkinElmer Life Sciences). Sections were incubated with a 1:100 dilution of APC-labeled anti-mouse CD11b (clone M1/70), FITClabeled anti-mouse E-cadherin, and PE-labeled anti-mouse CD11c (HM3) at 4C for 1 h (antibody dilutions were performed in PBS containing 1:200 Fc Block). Normal rabbit IgG (Santa Cruz Biotechnology.) was used as an isotype control. All slides were mounted in ProLong antifade medium (Molecular Probes). Images were acquired using a Zeiss LSM510 confocal microscope and image editing was done with Photoshop software (Adobe) and/or Spot Advanced software (Diagnostic Instruments). E-cadherin staining was pseudocolored in blue and CD11c and CD4 stainings were pseudocolored in red to enhance signal clarity in printed images.
4 Supplementary Figure 1 Phenotype of LP macrophages. Cells isolated from spleen and small intestine LP were stained with indicated antibodies and analyzed by flow cytometry. Pre-gated CD11b + CD11c /lo cells are shown. Solid black lines indicate staining with isotype control antibodies and shaded histograms represent specific stains. Data are representative of one of three independent experiments.
5 Supplementary Figure 2 LP macrophages express an anti-inflammatory gene expression signature. RNA was isolated from sorted splenic or intestinal CD11b + CD11c /dull cells, and expression of Il10, Tgfb1, and Tgfb3 was analyzed by quantitative real-time PCR. Values are expressed relative to Gapdh. Data are representative of one of two independent experiments.
6 Supplementary Figure 3 Effect of IL-10 on splenic or LP macrophage-induced T cell cytokine responses. Intestine (a) and spleen (b) CD11b + CD11c cells purified from Il10 +/+ or Il10 / B6 mice were cultured in with or without antibodies neutralizing IL-10 and IL- 10R in media alone ( ), E. coli lipopolysacchride (LPS; 1µg/ml), Pam3Cys (Pam; 10 µg/ml), yeast zymosan (Zymo; 10 µg/ml), or (CpG; 1µg/ml). After 24 h IL-12p40, and IL-12p70 in supernatants were measured by ELISA. Data are representative of one of two independent experiments.
7 Supplementary Figure 4 LP macrophages inhibit T H 1 polarization of CD4 + T cells. (a) B6.PL (Thy1.1 + ) recipient mice were reconstituted with OT-II T cells (Thy1.2 + ) one day prior to receiving OVA- or medium-pulsed splenic (spleen) or LP (intestine) CD11b + cells in the footpad. Four days later, the presence of transferred OT-II cells in draining lymph nodes was measured by flow cytometry. Data are representative of one of three independent experiments. (b) Draining lymph node cells were restimulated in vitro with OVA for 48 h and cytokines were measured by ELISA. *P < 0.05
8 Supplementary Figure 5 Retinoic acid is required for efficient generation of FoxP3 + T reg cells. (a) RNA was isolated from purified splenic or LP CD11b + cells, and expression of Aldh1a1 and Aldh1a2 were analyzed by quantitative real-time PCR. Values are expressed relative to Gapdh. (b) OT-II T cells were stimulated with LP macrophages for 4 days with OVA and TGF-β in the absence ( ) or presence of the RA receptor antagonist LE540. T reg cell differentiation was analyzed by intracellular staining and flow cytometry. Data are representative of one of two independent experiments.
9 Supplementary Figure 6 IL-10 does not inhibit IL-17 production induced by intestinal DCs. OT-II T cells were co-cultured with intestine DCs and OVA for 4 days in the absence ( ) or presence of IL-10 and were subsequently restimulated with plate-bound anti-cd3 and anti-cd28 for 6 h. Intracellular IL-17 and IFN-γ production was assayed by flow cytometry. Numbers indicate percentages of cells within quadrants. Data are representative of one of two independent experiments.
10 Supplementary Figure 7 In vivo depletion of LP DCs. CD11c-DTR mice were treated orally with diphtheria toxin for 10 days (every two days). (a) On day 10, intestines were removed and isolated cells were stained with indicated antibodies and analyzed by flow cytometry. Data are representative of one of three independent experiments. (b) On day 10, purified LP lymphocytes were stimulated in vitro with PMA and ionomycin for 5 h and subjected to intracellular staining for IL-17 (top) or left unstimulated and subjected to intracellular staining for FoxP3 (bottom). Pre-gated TCRβ + CD4 + cells are shown. Data are representative of one of three independent experiments.
11 Supplementary Figure 8 Model of APC function in the LP.
12 Supplementary Table 1. Real-time PCR Primers. Gene Name Gene Sequence (5-3 ) Gapdh Sense Gapdh Antisense Tgfb1 Sense Tgfb1 Antisense Tgfb3 Sense Tgfb3 Antisense Il10 Sense Il10 Antisense Aldh1a1 Sense Aldh1a1 Antisense Aldh1a2 Sense Aldh1a2 Antisense TGGCAAAGTGGAGATTGTTGCC AAGATGGTGATGGGCTTCCCG ACCATGCCAACTTCTGTCTG CGGGTTGTGTTGGTTGTAGA GGAAATGGGTCCACGAACCTA TCCAAGCACCGTGCTATGG CCCTTTGCTATGGTGTCCTT TGGTTTCTCTTCCCAAGACC ATGGTTTAGCAGCAGGACTCTTC CCAGACATCTTGAATCCACCGAA GACTTGTAGCAGCTGTCTTCACT TCACCCATTTCTCTCCCATTTCC
SI Appendix. Tumor-specific CD8 + Tc9 cells are superior effector than Tc1 cells for. adoptive immunotherapy of cancers
SI Appendix Tumor-specific CD8 + Tc9 cells are superior effector than Tc1 cells for adoptive immunotherapy of cancers Yong Lu, Bangxing Hong, Haiyan Li, Yuhuan Zheng, Mingjun Zhang, Siqing Wang, Jianfei
More informationSupplementary Figure 1. Phenotype, morphology and distribution of embryonic GFP +
Supplementary Figure 1 Supplementary Figure 1. Phenotype, morphology and distribution of embryonic GFP + haematopoietic cells in the intestine. GFP + cells from E15.5 intestines were obtained after collagenase
More informationTable S1. Antibodies and recombinant proteins used in this study
Table S1. Antibodies and recombinant proteins used in this study Labeled Antibody Clone Cat. no. Streptavidin-PerCP BD Biosciences 554064 Biotin anti-mouse CD25 7D4 BD Biosciences 553070 PE anti-mouse
More informationAutocrine Complement Inhibits IL10-Dependent T-Cell Mediated. Antitumor Immunity to Promote Tumor Progression
Supplemental Information Autocrine Complement Inhibits IL10-Dependent T-Cell Mediated Antitumor Immunity to Promote Tumor Progression Yu Wang, Sheng-Nan Sun, Qing Liu, Yang-Yang Yu, Jian Guo, Kun Wang,
More informationSupplementary Methods
Supplementary Methods Mice injections. C57BL/6 female mice 6-10 weeks of age were purchased from the Jackson Laboratory. Soluble rapamycin (Sigma) was diluted in PBS and administered i.p. to mice at 1.5
More informationSupplementary Figure 1. Characterization of the POP2 transcriptional and post-transcriptional regulatory elements. (A) POP2 nucleotide sequence
1 5 6 7 8 9 10 11 1 1 1 Supplementary Figure 1. Characterization of the POP transcriptional and post-transcriptional regulatory elements. (A) POP nucleotide sequence depicting the consensus sequence for
More informationSupplementary Figure 1
Supplementary Figure 1 Ex2 promotor region Cre IRES cherry pa Ex4 Ex5 Ex1 untranslated Ex3 Ex5 untranslated EYFP pa Rosa26 STOP loxp loxp Cre recombinase EYFP pa Rosa26 loxp 1 kb Interleukin-9 fate reporter
More informationThe RT-qPCR analysis of selected type-i IFNs related genes, IRF7 and Oas3. The
SUPPLEMENTARY MATERIALS AND METHODS Real time quantitative PCR The RT-qPCR analysis of selected type-i IFNs related genes, IRF7 and Oas3. The RT-qPCR was performed on the Applied Biosystems StepOne TM
More informationHuman skin punch biopsies were obtained under informed consent from normal healthy
SUPPLEMENTAL METHODS Acquisition of human skin specimens. Human skin punch biopsies were obtained under informed consent from normal healthy volunteers (n = 30) and psoriasis patients (n = 45) under a
More informationisolated from ctr and pictreated mice. Activation of effector CD4 +
Supplementary Figure 1 Bystander inflammation conditioned T reg cells have normal functional suppressive activity and ex vivo phenotype. WT Balb/c mice were treated with polyi:c (pic) or PBS (ctr) via
More informationSupplementary figures
Mucida et al. Supplementary material Supplementary figures Supplementary Figure 1. Oral administration of OVA suppresses Th2 differentiation, Germinal Center (GC) formation and immunoglobulin class switching
More informationMeasurement of peritoneal macrophage apoptosis by Celigo plate imaging cytometer
SUPPLEMENTAL METHODS Measurement of peritoneal macrophage apoptosis by Celigo plate imaging cytometer For Celigo experiments, 0.1 ml containing 5 x 10 4 cells was seeded into 96 well plates for 30 min
More informationPITT ISL METHODS 1) Flow-based CTL assay 2) Gut Mucosal Processing
PITT ISL METHODS 1) Flow-based CTL assay a. Purified CD8 + T cells are co-cultured with fresh, autologous, infected CD4 + T cells at various effector/target ratios for 18 hours at 37 o C. b. Baseline percentage
More informationMayumi Egawa, Kaori Mukai, Soichiro Yoshikawa, Misako Iki, Naofumi Mukaida, Yohei Kawano, Yoshiyuki Minegishi, and Hajime Karasuyama
Immunity, Volume 38 Supplemental Information Inflammatory Monocytes Recruited to Allergic Skin Acquire an Anti-inflammatory M2 Phenotype via Basophil-Derived Interleukin-4 Mayumi Egawa, Kaori Mukai, Soichiro
More informationSupplemental figure 1: Phenotype of IMC and MDSC after purification. A. Gating
Supplemental Figure Legend: Supplemental figure 1: Phenotype of IMC and MDSC after purification. A. Gating strategy for mouse MDSC. CD11b + Ly6C high Ly6G - cells are defined as M-MDSC. CD11b + Ly6C low
More informationClustalW algorithm. The alignment statistics are 96.9% for both pairwise identity and percent identical sites.
Identity Human Mouse Supplemental Figure 1. Amino acid sequence alignment of -MyHC. The alignment was created using the ClustalW algorithm. The alignment statistics are 96.9% for both pairwise identity
More informationDendritic epidermal T cells regulate skin antimicrobial barrier function
Online supplemental material Dendritic epidermal T cells regulate skin antimicrobial barrier function Amanda S. MacLeod 1, Saskia Hemmers 1,2, Olivia Garijo 1, Marianne Chabod 1, Kerri Mowen 1,2, Deborah
More informationNature Biotechnology: doi: /nbt.4086
Ag (-) anti-cd3 p815 p815-hcd2 Ag (-) anti-cd3 p815 p815-hcd2 Ag (-) anti-cd3 p815 p815-hcd2 Ag (-) anti-cd3 p815 p815-hcd2 Ag (-) anti-cd3 p815 p815-hcd2 Ag (-) anti-cd3 p815 p815-hcd2 Ag (-) anti-cd3
More informationThe following fluorochrome-conjugated antibodies were used: FITC and Alexa Fluor 700
Flow cytometry analysis and cell sorting The following fluorochrome-conjugated antibodies were used: FITC and Alexa Fluor 700 anti-cd5. (), APC-Cy7 anti-epcam (G.; Biolegend, San Diego, CA), PE-Cy7 anti-cd31
More informationSupplemental methods Supplemental figure and legend...7. Supplemental table.. 8
Supplemental Digital Content (SDC) Contents Supplemental methods..2-6 Supplemental figure and legend...7 Supplemental table.. 8 1 SDC, Supplemental Methods Flow cytometric analysis of intracellular phosphorylated
More informationby Alexander Y. Rudensky (Sloan-Kettering Institute, New York). LSL-TβRI CA (TGFβR-
Materials and Methods Mice. Tgfbr2 fl/fl CD4-Cre (TGFβR-KO) mice were previously reported (3). Cre negative littermates were used as wild-type controls. DN-TGFβRII (TGFβR-DN) mice were provided by Ronald
More informationSupplementary Figure 1. Effect of FRC-specific ablation of Myd88 on PP and mln organization.
Supplementary Figure 1 Effect of FRC-specific ablation of Myd88 on PP and mln organization. (a) PP numbers in 8 10 week old Cre-negative littermate (Ctrl) and Myd88-cKO mice (n = 11 mice; each dot represents
More informationSupplemental Materials and Methods
Supplemental Materials and Methods 125 I-CXCL12 binding assay KG1 cells (2 10 6 ) were preincubated on ice with cold CXCL12 (1.6µg/mL corresponding to 200nM), CXCL11 (1.66µg/mL corresponding to 200nM),
More informationB Vehicle 1V270 (35 μg) 1V270 (100 μg) Days post tumor implantation. Vehicle 100μg 1V270 biweekly 100μg 1V270 daily
Supplemental Figure 1 A 1 1V7 (8 μg) 1 1V7 (16 μg) 8 6 4 B 1 1 8 6 4 1V7 (35 μg) 1V7 (1 μg) 5 1 15 5 1 15 C Biweekly 8 11 14 17 Days Implant SCC7 cells Daily 8 9 1 11 1 Implant SCC7 cells 1V7 i.t. treatment
More informationSupplemental Information Inventory
Cell Stem Cell, Volume 6 Supplemental Information Distinct Hematopoietic Stem Cell Subtypes Are Differentially Regulated by TGF-β1 Grant A. Challen, Nathan C. Boles, Stuart M. Chambers, and Margaret A.
More informationFemale 6-7-week-old BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan).
Supplementary materials Materials and Methods Mice Female 6-7-week-old BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan). All mice were maintained under specific pathogen free conditions, and
More informationSupporting Information
Supporting Information Cieslewicz et al. 10.1073/pnas.1312197110 SI Results Human and mouse lesions of atherosclerosis contain both M1 and M2 macrophage phenotypes (1, 2). Previous work has suggested the
More informationSupplemental Information The Sensing of Environmental Stimuli by Follicular Dendritic Cells Promotes Immunoglobulin A Generation in the Gut
Immunity, Volume 33 Supplemental Information The Sensing of Environmental Stimuli by Follicular Dendritic Cells Promotes Immunoglobulin A Generation in the Gut Keiichiro Suzuki, Mikako Maruya, Shimpei
More informationCD1a-autoreactive T cells are a normal component of the human αβ T cell repertoire
CDa-autoreactive T cells are a normal component of the human αβ T cell repertoire Annemieke de Jong, Victor Peña-Cruz, Tan-Yun Cheng, Rachael A. Clark, Ildiko Van Rhijn,, D. Branch Moody Division of Rheumatology,
More informationSupplementary Figure 1: Analysis of monocyte subsets and lineage relationships. (a) Gating strategy for definition of MDP and cmop populations in BM
Supplementary Figure 1: Analysis of monocyte subsets and lineage relationships. (a) Gating strategy for definition of MDP and cmop populations in BM of Cx3cr1 GFP/+ mice related to Fig. 1a. MDP was defined
More informationNature Medicine doi: /nm.3554
SUPPLEMENTARY FIGURES LEGENDS Supplementary Figure 1: Generation, purification and characterization of recombinant mouse IL-35 (ril-35). High-Five insect cells expressing high levels of the bicistronic
More informationSingle-cell suspensions of C57BL/6J splenocytes were incubated with CD5 beads
S S Single-cell suspensions of C57BL/6J splenocytes were incubated with CD5 beads (Miltenyi Biotech) and T cells were positively selected by magnetically activated cell sorting (MACS). 50x10 6 or greater
More informationSupplementary methods. RNA isolation, cdna synthesis, and quantitative real-time PCR. Total RNAs were
Supplementary methods RNA isolation, cdna synthesis, and quantitative real-time PCR. Total RNAs were extracted from cells by using an RNeasy Mini Kit (QIAGEN) and then reverse-transcribed using the SuperScript
More informationFlow cytometry Stained cells were analyzed and sorted by SORP FACS Aria (BD Biosciences).
Mice C57BL/6-Ly5.1 or -Ly5.2 congenic mice were used for LSK transduction and competitive repopulation assays. Animal care was in accordance with the guidelines of Keio University for animal and recombinant
More informationSUPPLEMENTARY INFORMATION
doi:10.1038/nature10772 Supplementary Figures: Supplementary Figure 1. Location of CNS1 within of the Foxp3 locus highlighting CNS1 and indicating transcription factor binding motifs downstream of TCR,
More informationEstablishing sirna assays in primary human peripheral blood lymphocytes
page 1 of 7 Establishing sirna assays in primary human peripheral blood lymphocytes This protocol is designed to establish sirna assays in primary human peripheral blood lymphocytes using the Nucleofector
More informationThe presence of T cell epitopes is important for induction of antibody
The presence of T cell epitopes is important for induction of antibody responses against antigens directed to DEC25 + dendritic cells Kelly N. S. Amorim, Eline V. Rampazo, Renan Antonialli, Marcio M. Yamamoto,
More informationImmunofluorescent staining and flow cytometric analysis of cells
UCAN-U 0003 Version 1 November 2010 Page 1 of 7 CMCI (Center for molecular and Cellular Intervention) University Medical Center Utrecht Written By Name Function Date Signature Mark Klein Lab manager Conformation
More informationEnumeration, Phenotyping, and Identification of Activation Events in Conjugates Between T Cells and Antigen-Presenting Cells by Flow Cytometry
Enumeration, Phenotyping, and Identification of Activation Events in Conjugates Between T Cells and Antigen-Presenting Cells by Flow Cytometry Kristie M. Grebe and Terry A. Potter* (Published 10 September
More informationSupplementary Appendix
Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Svegliati Baroni S, Santillo M, Bevilacqua F, et al. Stimulatory
More informationSupporting Information
Supporting Information of Laser Immunotherapy in Combination with Perdurable PD-1 Blocking for Treatment of Metastatic Tumor Lihua Luo, Chunqi Zhu, Hang Yin, Mengshi Jiang, Junlei Zhang, Bing Qin, Zhenyu
More information15h. 24h. Blander & Medzhitov supplementary Figure 1. Apoptotic cells. Apoptotic LPS blasts 30% 32% 32% + Exogenous LPS 0.1% 31% 21% 20% 48% 60% 53%
a None Apoptotic cells Apoptotic LPS blasts 30% 32% 32% Apoptotic cells + Exogenous LPS 6h 0.1% 31% 21% 20% 48% 60% 53% 15h 0% 7% 5% 4% 30% 32% 32% 24h CD11c 0% 8% 2% 2% CFSE Blander & Medzhitov supplementary
More informationWhole Spleen Flow Cytometry Assay Cathy S. Yam *, Adeline M. Hajjar *
Whole Spleen Flow Cytometry Assay Cathy S. Yam *, Adeline M. Hajjar * Department of Comparative Medicine, University of Washington, Seattle, USA *For correspondence: csyam@u.washington.edu; hajjar@uw.edu
More informationSUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION ARTICLE NUMBER: 16108 DOI: 10.1038/NMICROBIOL.2016.108 The binary toxin CDT enhances Clostridium difficile virulence by suppressing protective colonic eosinophilia Carrie A. Cowardin,
More informationSTING-Dependent Cytosolic DNA Sensing Promotes Radiation-Induced Type I Interferon-Dependent Antitumor Immunity in Immunogenic Tumors
Immunity, Volume 41 Supplemental Information STING-Dependent Cytosolic DNA Sensing Promotes Radiation-Induced Type I Interferon-Dependent Antitumor Immunity in Immunogenic Tumors Liufu Deng, Hua Liang,
More informationSegments of the obstructed intestinal loops were fixed in 4% paraformaldehyde
Supplementary text Supplementary materials and methods Histopathological examination Segments of the obstructed intestinal loops were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin wax with
More informationNature Immunology: doi: /ni Supplementary Figure 1. Time course of OT-II T reg cell development in thymic slices.
Supplementary Figure 1 Time course of OT-II T reg cell development in thymic slices. Time course of T reg cell development following addition of OT-II TCR transgenic Rag2 -/- mice (OT-II) thymocytes to
More informationSupplementary Figure 1. Gating strategy for flow cytometry analysis of mouse aorta. Cell suspensions from mouse aorta digested with enzyme cocktail
Supplementary Figure 1. Gating strategy for flow cytometry analysis of mouse aorta. Cell suspensions from mouse aorta digested with enzyme cocktail were stained with propidium iodide (PI), anti-cd45 (FITC),
More informationNanogel-Based Immunologically Stealth Vaccine Targets Macrophages in the Medulla of Lymph Node and Induces Potent Antitumor Immunity
Nanogel-Based Immunologically Stealth Vaccine Targets Macrophages in the Medulla of Lymph Node and Induces Potent Antitumor Immunity Daisuke Muraoka, Naozumi Harada,, Tae Hayashi, Yoshiro Tahara,, Fumiyasu
More informationFigure S1. Phenotypic characterization of AND-1_WASKO cell lines. AND- 1_WASKO_C1.1 (WASKO_C1.1) and AND-1_WASKO_C1.2 (WASKO_C1.
LEGENDS TO SUPPLEMENTARY FIGURES Figure S1. Phenotypic characterization of AND-1_WASKO cell lines. AND- 1_WASKO_C1.1 (WASKO_C1.1) and AND-1_WASKO_C1.2 (WASKO_C1.2) were stained with the antibodies oct3/4
More informationSupporting Information for. Bongseo Choi, 1, Hyojin Moon, 1, Sung Joon Hong, 1 Changsik Shin, 1 Yoonkyung Do, 1 Seongho Ryu, 2,* Sebyung Kang 1,*
Supporting Information for Effective Delivery of Antigen-Encapsulin Nanoparticle Fusions to Dendritic Cells Leads to Antigen-Specific Cytotoxic T Cell Activation and Tumor Rejection Bongseo Choi, 1, Hyojin
More informationCFSE Cell Division Assay Kit
CFSE Cell Division Assay Kit Item No. 10009853 www.caymanchem.com Customer Service 800.364.9897 Technical Support 888.526.5351 1180 E. Ellsworth Rd Ann Arbor, MI USA TABLE OF CONTENTS GENERAL INFORMATION
More informationSupporting Information
Supporting Information Narni-Mancinelli et al. 10.1073/pnas.1112064108 SI Materials and Methods Cell Preparation. Mice were anesthetized and immediately perfused with PBS before organs were collected.
More informationBD Pharmingen. Mouse Th1/Th2/Th17 Phenotyping Kit. Technical Data Sheet. Product Information. Description Components:
BD Pharmingen Technical Data Sheet Mouse Th1/Th2/Th17 Phenotyping Kit Product Information Material Number: 560758 Size: 50 tests Description Components: 51-9006631 Mouse Th1/Th2/Th17 Phenotyping Cocktail
More informationThe transcrip-on factor NR4A1 (Nur77) controls bone marrow differen-a-on and survival of Ly6C monocytes
Supplementary Informa0on The transcrip-on factor NR4A1 (Nur77) controls bone marrow differen-a-on and survival of Ly6C monocytes Richard N. Hanna 1, Leo M. Carlin 2, Harper G. Hubbeling 3, Dominika Nackiewicz
More informationSUnSET, a nonradioactive method to monitor protein synthesis
nature methods SUnSET, a nonradioactive method to monitor protein synthesis Enrico K Schmidt, Giovanna Clavarino, Maurizio Ceppi & Philippe Pierre Supplementary figures and text: Supplementary Figure 1
More informationIn vivo BrdU Incorporation Assay for Murine Hematopioetic Stem Cells Ningfei An, Yubin Kang *
In vivo BrdU Incorporation Assay for Murine Hematopioetic Stem Cells Ningfei An, Yubin Kang * Division of Hematology-Oncology, Department of Medicine, Medical University of South Carolina, Charleston,
More informationSUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION doi:10.1038/nature11809 Supplementary Figure 1. Antibiotic treatment reduces intestinal bacterial load and allows access of non-pathogenic bacteria to the MLN, inducing intestinal
More informationFigure S1 Proteasome inhibition leads to formation of aggregates in human cells and tissues. (a)
SUPPLEMENTARY MATERIAL Figure S1 Proteasome inhibition leads to formation of aggregates in human cells and tissues. (a) Flow cytometry. Cells were treated with MG132 for the indicated times. Cells were
More informationSupplementary Information (Ha, et. al) Supplementary Figures Supplementary Fig. S1
Supplementary Information (Ha, et. al) Supplementary Figures Supplementary Fig. S1 a His-ORMDL3 ~ 17 His-ORMDL3 GST-ORMDL3 - + - + IPTG GST-ORMDL3 ~ b Integrated Density (ORMDL3/ -actin) 0.4 0.3 0.2 0.1
More informationefluor Organic Dyes 450/50 BP Fluorescence Intensity Wavelength (nm)
efluor Organic Dyes efluor Organic Dyes Catalog No. Description Clone Application Anti-Mouse efluor 450 Products 48-0042 Anti-mouse CD4 RM4-5 Flow Cytometry 48-0081 Anti-mouse CD8 53-6.7 Flow Cytometry
More informationBD Biosciences BD Cytometric Bead Array (CBA) Product List. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD Biosciences BD Cytometric Bead Array (CBA) Product List For Research Use Only. Not for use in diagnostic or therapeutic procedures. Highlights of BD CBA Products Reagents with Superior Quality and Reproducibility
More informationGene Sequence Annealing Temperature. Actin 5 : 5 -CATCACTATTGGCAACGAGC-3 60 C 3 : 5 -ACGCAGCTCAGTAACAGTCC-3 PCR product: 410 bp
Supporting Materials and Methods RT-PCR RT-PCR was performed as described in ref. 1 using the following oligonucleotides and PCR conditions: 2 min at 95 C, (20 s at 95 C, 20 s at the annealing temp, and
More informationNature Immunology: doi: /ni Supplementary Figure 1. Zranb1 gene targeting.
Supplementary Figure 1 Zranb1 gene targeting. (a) Schematic picture of Zranb1 gene targeting using an FRT-LoxP vector, showing the first 6 exons of Zranb1 gene (exons 7-9 are not shown). Targeted mice
More informationInterferon- -producing immature myeloid cells confer protection. against severe invasive group A Streptococcus infections. Supplementary Information
Supplementary Information Interferon- -producing immature myeloid cells confer protection against severe invasive group A Streptococcus infections Takayuki Matsumura, Manabu Ato, Tadayoshi Ikebe, Makoto
More informationLife Sciences Reporting Summary
Life Sciences Reporting Summary Corresponding Author: Date: Wanjun Chen & Keji Zhao Nature Research wishes to improve the reproducibility of the work we publish. This form is published with all life science
More informationSupplementary Materials for
www.sciencesignaling.org/cgi/content/full/1/494/eaak972/dc1 Supplementary Materials for Blockade of surface-bound TGF-β on regulatory T cells abrogates suppression of effector T cell function in the tumor
More informationSupplementary Information for Transient and Local Expression of Chemokine and Immune Checkpoint Traps to Treat Pancreatic Cancer
Supplementary Information for Transient and Local Expression of Chemokine and Immune Checkpoint Traps to Treat Pancreatic Cancer Lei Miao 1, Jingjing Li 2, Qi Liu 1,4, Richard Feng 2, Manisit Das 1, C.
More informationSupplemental Materials and Methods
Supplemental Materials and Methods In situ hybridization In situ hybridization analysis of HFE2 and genin mrna in rat liver tissues was performed as previously described (1). Briefly, the digoxigenin-labeled
More informationGeneration of mouse BMDCs Vectors Short hairpin RNA constructs cdna constructs and generation of stable cell lines
Generation of mouse BMDCs Mouse BMDCs were differentiated from BM cells isolated from femur and tibiae of the hind legs. Cell suspensions were filtered through a 40 µm cell strainer and incubated for 2
More informationMice TRAMP mice were maintained in a C57BL/6J background. Syngeneic UBI-GFP/BL6 mice were used for bone marrow engraftment. 2
Antibodies Chicken IgY polyclonal α-gfp antibodies were purchased from Abcam (Cambridge, MA) and were detected using α-chicken IgY-FITC or α-chicken-hrp (also purchased from Abcam). The CD31-PE, CD11b-PE,
More informationTo determine the effect of N1IC in the susceptibility of T cells to the tolerogenic effect of tumorassociated
Supplementary Methods Tolerogenic effect of MDSC To determine the effect of N1IC in the susceptibility of T cells to the tolerogenic effect of tumorassociated MDSC in vivo, we used a model described previously
More informationSupplementary Figure. S1
Supplementary Figure. S1 Supplementary Figure S1. Correlation of phagocytic ability measured with YG and YO beads. Fresh human monocytes (2 10 6 /ml) were labelled with APC conjugated anti CD14 mab alone
More informationa Lamtor1 (gene) b Lamtor1 (mrna) c WT Lamtor1 Lamtor1 flox Lamtor2 A.U. p = LysM-Cre Lamtor3 Lamtor4 Lamtor5 BMDMs: Φ WT Φ KO β-actin WT BMDMs
a Lamtor (gene) b Lamtor (mrna) c BMDMs: Φ WT Φ KO Lamtor flox 8 bp LysM-Cre 93 bp..5 p =.4 WT BMDMs: Φ WT Φ KO Lamtor (protein) BMDMs: Φ WT Φ KO Lamtor 8 kda Lamtor 4 kda Lamtor3 4 kda Lamtor4 kda Lamtor5.5
More informationTitle: Stromal Cell Subsets Directing Neonatal Spleen Regeneration
Title: Stromal Cell Subsets Directing Neonatal Spleen Regeneration Authors: Jonathan K.H. Tan and Takeshi Watanabe SUPPLEMENTAL INFORMATION Supplemental Tables 1-4 Supplemental Figure 1 Table S1. Marker
More informationDC enriched CD103. CD11b. CD11c. Spleen. DC enriched. CD11c. DC enriched. CD11c MLN
CD CD11 + CD + CD11 d g CD CD11 + CD + CD11 CD + CD11 + Events (% of max) Events (% of max) CD + CD11 Events (% of max) Spleen CLN MLN Irf fl/fl e Irf fl/fl h Irf fl/fl DC enriched CD CD11 Spleen DC enriched
More informationWelcome to More Choice. Mouse Panels
Welcome to More Choice Mouse Panels Choose from our extensive portfolio of high-quality fluorescent-conjugated reagents to build your multicolor flow cytometry panels. Welcome to a More Colorful World
More informationPE11, a PE/PPE family protein of Mycobacterium tuberculosis is involved in cell wall remodeling and virulence
PE11, a PE/PPE family protein of Mycobacterium tuberculosis is involved in cell wall remodeling and virulence Parul Singh 1,2, Rameshwaram Nagender Rao 1, Jala Ram Chandra Reddy 3, R.B.N. Prasad 3, Sandeep
More informationSupplementary Information
Supplementary Information Mutual reinforcement of inflammation and carcinogenesis by the Helicobacter pylori CagA oncoprotein Nobumi Suzuki, Naoko Murata-Kamiya, Kohei Yanagiya, Wataru Suda, Masahira Hattori,
More informationA collection of 31 splenic specimens from patients (42.4±17.7 years old) with ITP
Supplemental Materials & Methods Patients and patient samples. A collection of 31 splenic specimens from patients (42.4±17.7 years old) with ITP requiring splenectomy and 36 splenic specimens obtained
More informationIL-17 Regulates Systemic Fungal Immunity by Controlling the Functional Competence of NK Cells
Immunity, Volume 40 Supplemental Information IL-17 Regulates Systemic Fungal Immunity by Controlling the Functional Competence of NK Cells Eva Bär, Paul G. Whitney, Kathrin Moor, Caetano Reis e Sousa,
More informationSupplementary Figure 1 - Characterization of rbag3 binding on macrophages cell surface.
Supplementary Figure 1 - Characterization of rbag3 binding on macrophages cell surface. (a) Human PDAC cell lines were treated as indicated in Figure 1 panel F. Cells were analyzed for FITC-rBAG3 binding
More informationAntibody Titrations. Institut für HIV Forschung SOP #06-03 (Nov BS) Background. Reagents
Institut für HIV Forschung SOP #06-03 (Nov 2017 - BS) Antibody Titrations Reagents Reagent Vendor Catalogue # Stock Conc. FACS Tubes BD Falcon 352054 - Anti-CD28/CD49d Antibody BD Fastimmune 347690 - GolgiPlug
More informationFlow-cytometry assessment of parasitemia in cell parasite co-culture assay using hydroethidine staining Western blotting RT-PCR
Flow-cytometry assessment of parasitemia in cell parasite co-culture assay using hydroethidine staining The capability of cells, either PBMC or purified T- cell lines, to inhibit parasite growth or merozoite
More informationSANTA CRUZ BIOTECHNOLOGY, INC.
TECHNICAL SERVICE GUIDE: Western Blotting 2. What size bands were expected and what size bands were detected? 3. Was the blot blank or was a dark background or non-specific bands seen? 4. Did this same
More informationPage 1 of 2. Product Information Contents: ebioscience BrdU Staining Kit for Flow Cytometry APC
Page 1 of 2 ebioscience BrdU Staining Kit for Flow Cytometry APC Also known as: 5-bromodeoxyuridine RUO: For Research Use Only. Not for use in diagnostic procedures. Anti-CD3/CD28-stimulated mouse splenocytes
More informationPage 1 of 2. Product Information Contents: ebioscience BrdU Staining Kit for Flow Cytometry FITC
Page 1 of 2 ebioscience BrdU Staining Kit for Flow Cytometry FITC Also known as: 5-bromodeoxyuridine RUO: For Research Use Only. Not for use in diagnostic procedures. Anti-CD3/CD28-stimulated mouse splenocytes
More informationSupplementary Figure 1. Lack of autoimmunity in NKT cell deficient B6.NZBc4L
Supplementary Figure. Lack of autoimmunity in NKT cell deficient L mice. A,.CDd-deficient mice lack CDd-independent NKT cells. B, In the absence of NKT cells,.cdd -/- mice fail to generate high titres
More informationSupplementary Methods
Supplementary Methods Generation of CX3CR1-GFP KI Mice Briefly, four individual DNA segments, including murine 3 -flanking region, neomycin resistance gene flanked by loxp sites, hcx 3 CR1-GFP, and murine
More informationDevelopment of CD11b-IRF8 transgenic and MTAG double transgenic mice The CMV-
Supplemental Data Development of CD11b-IRF8 transgenic and MTAG double transgenic mice The CMV- IRF8 transgenic mouse was previously developed by cloning complementary DNA, containing the IRF-8 coding
More informationSupplementary Materials for
advances.sciencemag.org/cgi/content/full/4/9/eaat5401/dc1 Supplementary Materials for GLK-IKKβ signaling induces dimerization and translocation of the AhR-RORγt complex in IL-17A induction and autoimmune
More informationSupplementary Material & Methods
Supplementary Material & Methods Affymetrix micro-array Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, USA). RNA concentration and quality was determined using the ND-1000 spectrophotometer
More informationM004 - Intracellular Cytokine Staining for Mouse Samples
Author Stephanie Swift Date 21st March 2017 Version 8 SOP # M004 Objective: This protocol addresses the processing, peptide-stimulation and antibody staining of fresh mouse samples for intracellular cytokine
More informationSupplemental Figure 1
Supplemental Figure 1 A IL-12p7 (pg/ml) 7 6 4 3 2 1 Medium then TLR ligands MDP then TLR ligands Medium then TLR ligands + MDP MDP then TLR ligands + MDP B IL-12p4 (ng/ml) 1.2 1..8.6.4.2. Medium MDP Medium
More informationMultiple layers of B cell memory with different effector functions. Ismail Dogan, Barbara Bertocci, Valérie Vilmont, Frédéric Delbos,
Multiple layers of B cell memory with different effector functions Ismail Dogan, Barbara Bertocci, Valérie Vilmont, Frédéric Delbos, Jérome Mégret, Sébastien Storck, Claude-Agnès Reynaud & Jean-Claude
More informationMurine and Non-Human Primate Dendritic Cell Targeting Nanoparticles for In Vivo Generation of Regulatory T-Cells
Murine and Non-Human Primate Dendritic Cell Targeting Nanoparticles for In Vivo Generation of Regulatory T-Cells Sebastian O. Stead 1, Svjetlana Kireta 2, Steven James Peter McInnes 3, Francis D. Kette
More informationlongitudinally and placed on a clean glass slide with luminal surface facing up. Epithelial cells
SUPPLEMENTARY MATERIALS AND METHODS: Protein extraction and Western blot Colons collected from mice were flushed with PBS. Each colon was then cut open longitudinally and placed on a clean glass slide
More informationSupplementary Materials & Methods
Supplementary Materials & Methods Mice and tumor lines. All experiments were performed using protocols approved by the University of Pennsylvania Laboratory Animal Resources (ULAR) policies and standard
More informationSUPPLEMENTARY INFORMATION
Supplementary Figure 1 (a) (b) Supplementary Figure 1. Histological difference between fat-associated lymphoid clusters (FALC) and lymph nodes. (a) H&E stained specimens of a mesenteric lymphoid cluster
More informationSupplementary Methods Antibodies for flow cytometry Cytotoxicity assay
Supplementary Methods Antibodies for flow cytometry HLA-Cw3 and CD2 expression: LCL 72.22 cell lines were stained with anti-hla-abc-apc (clone G46-2.6, BD) or migg-apc (clone MOPC-2, BD) antibody, or with
More information