Supplementary Table e-1. Flow cytometry reagents and staining combinations

Similar documents
SUPPLEMENTARY FIGURES

Categorical analysis of human T cell heterogeneity with One-SENSE

BD Flow Cytometry Reagents Multicolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-20 Cell Analyzer

Supplementary Materials

MAIT cell function is modulated by PD-1 signaling in patients with active

Supplementary Data. Treg phenotype

Fluorochrome Panel 1 Panel 2 Panel 3 Panel 4 Panel 5 CTLA-4 CTLA-4 CD15 CD3 FITC. Bio) PD-1 (MIH4, BD) ICOS (C398.4A, Biolegend) PD-L1 (MIH1, BD)

A step-by-step approach to build and analyze a multicolor panel

Supplementary Figure 1. Immune profiles of untreated and PD-1 blockade resistant EGFR and Kras mouse lung tumors (a) Total lung weight of untreated

CD25-PE (BD Biosciences) and labeled with anti-pe-microbeads (Miltenyi Biotec) for depletion of CD25 +

Supplementary Fig. 1: Ex vivo tetramer enrichment with anti-c-myc beads

Online supplement. Phenotypic, functional and plasticity features of classical and alternatively activated

Table S1. Viral load and CD4 count of HIV-infected patient population

Supplementalgfigureg1gSchematicgdiagramgofgtumor1modellingg

Recommended Protocols for Phospho Protein Detection in Human Cells

Optimizing Intracellular Flow Cytometry

Supplementary Figure 1. Enhanced detection of CTLA-4 on the surface of HIV-specific

Optimizing Intracellular Flow Cytometry:

IL-6Rα IL-6RαT-KO KO. IL-6Rα f/f bp. f/f 628 bp deleted 368 bp. 500 bp

Optimizing Intracellular Flow Cytometry:

Detailed step-by-step operating procedures for NK cell and CTL degranulation assays

Supporting Information

Commercially available HLA Class II tetramers (Beckman Coulter) conjugated to

Blocking antibodies and peptides. Rat anti-mouse PD-1 (29F.1A12, rat IgG2a, k), PD-

Comprehensive evaluation of human immune system reconstitution in NSG. and NSG -SGM3 mouse models toward the development of a novel ONCO-HU

Supplementary Figure 1. IL-12 serum levels and frequency of subsets in FL patients. (A) IL-12

Supplemental Information. Human CD1c + Dendritic Cells Drive. the Differentiation of CD103 + CD8 + Mucosal Effector T Cells via the Cytokine TGF-

Supplemental Table I.

SUPPORTING INFORMATIONS

Supplementary Figure 1. mtor LysM and Rictor LysM mice have normal cellularity and percentages of hematopoe>c cells. a. Cell numbers of lung, liver,

Glow. Save. Let it. and. Get a Discount of 10% BD Biosciences Year End Promotion on a huge Selection of Reagent Solutions

Naive, memory and regulatory T lymphocytes populations analysis

For surface staining, cells were stained with various markers (supplemental table S1) at room

- 1 - Cell types Monocytes THP-1 cells Macrophages. LPS Treatment time (Hour) IL-6 level (pg/ml)

Supplementary Figure S1. Flow cytometric analysis of the expression of Thy1 in NH cells. Flow cytometric analysis of the expression of T1/ST2 and

<10. IL-1β IL-6 TNF + _ TGF-β + IL-23

SUPPLEMENTARY INFORMATION. Involvement of IL-21 in the epidermal hyperplasia of psoriasis

CD4 + CD25 high Foxp3 + T regulatory cells kill autologous CD8(+) and CD4(+) T cells using Fas/FasL- and Granzyme B- mediated pathways

Table S1. New colony formation 7 days after stimulation with doxo and VCR in JURKAT cells

Fisher et al. Supplemental Figure 1

Cover Page. The handle holds various files of this Leiden University dissertation.

Interferon γ regulates idiopathic pneumonia syndrome, a. Th17 + CD4 + T-cell-mediated GvH disease

Supplementary Materials for

Supplementary Figure 1. BMS enhances human T cell activation in vitro in a

Supplementary Figure 1

Supplementary Information

L-selectin Is Essential for Delivery of Activated CD8 + T Cells to Virus-Infected Organs for Protective Immunity

Application Information Bulletin: Human NK Cells Phenotypic characterizing of human Natural Killer (NK) cell populations in peripheral blood

x Lymphocyte count /µl CD8+ count/µl 800 Calculated

The Annexin V Apoptosis Assay

PBMC from each patient were suspended in AIM V medium (Invitrogen) with 5% human

NK cell flow cytometric assay In vivo DC viability and migration assay

Combined Rho-kinase inhibition and immunogenic cell death triggers and propagates immunity against cancer

Supplementary Data 1. Alanine substitutions and position variants of APNCYGNIPL. Applied in

Pearson r = P (one-tailed) = n = 9

B6/COLODR/SPL/11C/83/LAP/#2.006 B6/COLODR/SPL/11C/86/LAP/#2.016 CD11C B6/COLODR/SPL/11C/80/LAP/#2.011 CD11C

HIV-infected subjects were recruited from an ongoing clinic-based cohort (SCOPE)

Supplementary Figure 1

Supplemental Material to:

Supplementary Information

The encephalitogenicity of TH17 cells is dependent on IL-1- and IL-23- induced production of the cytokine GM-CSF

DuraClone IM. Standardized phenotyping panels for studies of the human immune system

SUPPLEMENTARY INFORMATION

DIFFERENCES IN T-CELL SUBSETS IN ME/CFS. University of Nevada, Reno. Determining the Difference Between Circulating Follicular Regulatory T-cells and

SUPPLEMENT Supplementary Figure 1: (A) (B)

Supplemental Figure 1. IL-3 blockade with Fab CSL362 depletes plasmacytoid dendritic cells (pdcs), but not basophils, at higher doses.

Primary Adult Naïve CD4+ CD45RA+ Cells. Prepared by: David Randolph at University of Alabama, Birmingham

Figure S1. Analysis of genomic and cdna sequences of the targeted regions in WT-KI and

CELLULAR IMMUNE CORRELATES OF PROTECTION AGAINST

The Bcl-2 regulated apoptotic pathway is critical for the

BD Pharmingen. Human Th1/Th2/Th17 Phenotyping Kit. Technical Data Sheet. Product Information. Description Components:

Supplementary Information

Supplementary Figure 1. Ex vivo IFNγ production by Tregs. Nature Medicine doi: /nm % CD127. Empty SSC 98.79% CD25 CD45RA.

% of Cells A B C. Proliferation Index. T cell count (10 6 ) Division Index. % of Max CFSE. %Ki67+ cells. Supplementary Figure 1.

Supplementary Figure 1 Lymphocytes can be tracked for at least 4 weeks after

Kerdiles et al - Figure S1

Vaccine Delivery and TLR Ligands Influence the Quality of T cell Responses in NHP. Robert A. Seder, M.D. Vaccine Research Center NIAID, NIH

Supplemental Figure 1. Activated splenocytes upregulate Serpina3g and Serpina3f expression.

Contribution of microglia to tissue injury and repair in MS

Roles of calpain-calpastatin system (CCS) in human T cell activation

SUPPLEMENTARY METHODS

January 25, 2017 Scientific Research Process Name of Journal: ESPS Manuscript NO: Manuscript Type: Title: Authors: Correspondence to

ImageStream cytometer analysis. Cells were cultured as described above in vented-cap

Supplemental Methods In vitro T cell assays Inhibition of perforin- and FasL-mediated cytotoxicity Flow Cytometry

Supplementary Materials and Methods

ABSTRACT INTRODUCTION

Ex vivo Human Antigen-specific T Cell Proliferation and Degranulation Willemijn Hobo 1, Wieger Norde 1 and Harry Dolstra 2*

Human and mouse T cell regulation mediated by soluble CD52 interaction with Siglec-10. Esther Bandala-Sanchez, Yuxia Zhang, Simone Reinwald,

Nature Immunology: doi: /ni Supplementary Figure 1. Huwe1 has high expression in HSCs and is necessary for quiescence.

Supplemental Information. Human Circulating PD-1 + CXCR3 CXCR5 + Memory. Tfh Cells Are Highly Functional and Correlate

Nature Immunology: doi: /ni Supplementary Figure 1. Examples of staining for each antibody used for the mass cytometry analysis.

B and T-Cells. CD8+ Cytotoxic T Cells

Title. CitationCancer science, 109(4): Issue Date Doc URL. Rights(URL)

Supplementary Figure 1

Nature Protocols: doi: /nprot Supplementary Figure 1

of whole cell cultures in U-bottomed wells of a 96-well plate are shown. 2

Supplemental data. Antibodies used for Flow Cytometry. BL = Biolegend (San Diego, CA), BD = BD Biosciences (San Jose, CA) PD-1 Panel

Supplementary Figure S1. DD2 reactivity on human tonsils and analysis of slandc proliferation. Sections are from a representative human tonsil (a-f;

Alison M. McDonnell 1,2, Alistair Cook 1,2, Bruce W. S. Robinson 1,2,4, Richard A. Lake 1,2 and Anna K. Nowak 1,2,3*

BMDCs were generated in vitro from bone marrow cells cultured in 10 % RPMI supplemented

Transcription:

Supplementary data Supplementary Table e-1. Flow cytometry reagents and staining combinations Reagents Antibody Fluorochrome Clone Source conjugation CD3 FITC UCHT1 BD Biosciences CD3 PerCP-Cy5.5 SK7 Biolegend CD3 BUV395 SK7 BD Biosciences CD3 APC-R700 UCHT1 BD Biosciences CD4 BV786 SK3 BD Biosciences CD4 BUV661 SK3 BD Biosciences CD8 AlexaFluor700 RPA-T8 BD Biosciences CD8 BV786 RPA-T8 BD Biosciences CD45RA FITC HI100 BD Biosciences CD45RO PE UCHL1 BD Biosciences CD45RO APC-H7 UCHL1 BD Biosciences CCR7 APC 3D12 BD Biosciences CD31 PE WM59 BD Biosciences CD25 BV421 2A3 BD Biosciences CD127 PC7 R34.34 Beckman Coulter FoxP3 PE-CF594 236A/E7 BD Biosciences IFNγ BV650 4S.B3 BD Biosciences GM-CSF PE BVD2-21C11 BD Biosciences IL-10 APC JES3-19F1 BD Biosciences IL-17 BV421 N49-653 BD Biosciences IL-4 PE-CF594 MP4-25D2 BD Biosciences IL-22 PerCPeFluor710 22URTI ebioscience

Annexin V BUV395 N/A BD Biosciences Propidium iodide N/A N/A BD Biosciences Integrin β7 FITC FIB504 Biolegend CCR9 PE-Cy7 L053E8 Biolegend CCR5 BV421 J418F1 Biolegend CCR2 PerCP-Cy5.5 K036C2 Biolegend CLA PE HECA-452 BD Biosciences CCR4 PE-CF594 1G1 BD Biosciences BIM AF488 C34C5 Cell Signaling technology PUMA PE EP512Y Abcam BAX AF488 2D2 Biolegend BAK PE D4E4 Cell Signaling technology BCL-XL AF488 54H6 Cell Signaling technology BCL-2 BV421 100 Biolegend LIVE/DEAD Fixable N/A N/A Invitrogen Aqua Dead Cell Stain Staining combinations CD3-PerCP; CD4-BV786; CD8-AlexaFluor700; CD45RA-FITC; CCR7-APC; Live/Dead CD3-APC-R700; CD4-BV786; CD45RO-APC-H7; CD31-PE; CD25-BV421; CD127- PC7; FoxP3-PE-CF594 CD3-FITC; CD4-BV786; CD8-AlexaFluor700; CD45RO-PE; CCR7-APC; CD25- BV421; CD127-PC7; Annexin V-BUV395; Propidium iodide CD3-BUV395; CD4-BV786; CD8-AlexaFluor700; IFNγ-BV650; GM-CSF-PE; IL-10- APC; IL-17-BV421; IL-4-PE-CF594; IL-22-PerCP CD3-BUV395; CD4-BUV661; CD8-BV786; Integrin β7-fitc; CCR9-PE-Cy7; CCR5- BV421; CCR2-PerCP-Cy5.5; CLA-PE; CCR4-PE-CF594 CD3-BUV395; CD4-BUV661; CD8-BV786; BIM-AF4888; PUMA-PE CD3-BUV395; CD4-BUV661; CD8-BV786; BCL-XL-AF488; BCL-2-BV421 CD3-BUV395; CD4-BUV661; CD8-BV786; BAX-AF488; BAK-PE

Supplementary Figure e-1. T-cell subset staining and gating strategy Supplementary Figure e-1. T-cell subset staining and gating strategy. Representative example of T-cell subset staining by flow cytometry and gating strategy used from a MS patient sample.

Supplementary Figure e-2. Annexin V/propidium iodide staining Supplementary Figure e-2. Annexin V/propidium iodide staining. Representative example of Annexin/PI staining of CD8+ (top) and CD4+ (bottoom) T-cell subsets from a healthy control subject under each exposure. UNTX = medium alone, VEH = vehicle (DMSO), DMF = dimethyl fumarate, MMF = monomethyl fumarate, PI = propidium iodide.

Supplementary Figure e-3. DMF treatment in vivo alters the regulatory to effector cell balance Supplementary Figure e-3. DMF treatment in vivo alters the regulatory to effector cell balance. Greater losses in putatively pro-inflammatory effector T-cell populations versus putatively regulatory T-cell subsets were seen with DMF treatment of MS patients, leading to changes in regulatory to effector cell ratios. Increases were seen in the ratios of regulatory T-cells (Treg) to Th1 (A), Tc1 (B) and GM-CSFexpressing CD4+ T-cells (C), as well as in the ratios of IL-10-expressing CD4+ T- cells to Th1 (D) and GM-CSF-expressing CD4+ T cells (E). Data shown is from multiple sclerosis patients (n=13) pre-treatment (Month 0) and up to 12 months following DMF treatment initiation. The p values displayed represent the statistical significance of the exponential decay trajectory (shown in red) in a

random coefficient mixed effects model. Individual patient trajectories are shown in grey.

Supplementary Figure e-4. Susceptibility of circulating T-cells to dexamethasone-induced apoptosis pre- and post-dmf treatment Supplementary Figure e-4. Susceptibility of circulating T-cells to dexamethasone-induced apoptosis pre- and post-dmf treatment. Annexin V/PI staining to capture T-cell apoptosis following exposure to different concentrations of dexamethasone. Representative example of dose-dependent increase in dexamethasone-induced T-cell apoptosis (top row) compared to vehicle control (bottom row) using purified T-cells from a DMF-treated patient and gating on all CD3+ T-cells (A). Summary graphs showing frequencies of Annexin

V+/PI+ apoptotic cells (corrected for degree of apoptosis with vehicle control: Δ% An+/PI+ = % An+/PI+ with dexamethasone minus % An+PI+ with corresponding vehicle concentration) do not change significantly pre- versus on-treatment (n=3 patients; highest 3 concentrations of dexamethasone shown) (B). An = Annexin V, PI = propidium iodide, PRE = pre-treatment, ON = on-treatment.

Supplementary Table e-2. T-cell expression of pro- and anti-apoptotic molecules pre- and post-dmf treatment Molecules p value (pre- vs. posttreatment) Proapoptotic Antiapoptotic ΔMFI Pre-treatment (95% CI) ΔMFI Post-treatment (95% CI) BIM 191 (148-234) 188 (130-247) ns PUMA 2227 (501-3954) 2326 (1016-3635) ns BAX 152 (-54-357) 168 (-14-351) ns BAK 138 (115-161) 154 (121-187) ns BCL-XL 311 (245-377) 315 (200-429) ns BCL-2 2204 (704-3704) 2588 (971-4205) 0.0425 Supplementary Table e-2. T-cell expression of pro- and anti-apoptotic molecules pre- and post-dmf treatment. CD3+ T-cell expression of pro- and anti-apoptotic molecules pre- and post-in vivo treatment with DMF. Results are shown as mean ΔMFI (MFI of molecule minus MFI of isotype control) and 95% confidence intervals (n=3 matched pre-/posttreatment samples; p values compare pre- and post-treatment mean MFIs using paired t-tests). ns = not significant, MFI = mean fluorescence intensity.

Supplementary Figure e-5. Relative changes in tissue-homing T-cell populations with DMF treatment Supplementary Figure e-5. Relative changes in tissue-homing T-cell populations with DMF treatment. Representative example of staining for gut (CCR9+beta7integrin+), skin (CLA+CCR4+) and brain (CCR2+CCR5+) homing T-cell populations, gating on CD4+ cells (A). Summary graphs showing the change in relative frequency between matched pre- and 3 month post-treatment samples of each tissue homing population amongst CD4+ (B) and CD8+ (C) T-cells (n=3 patients; p values shown represent adjusted p values from repeated measures one-way ANOVA with Tukey s post test). DMF = dimethyl fumarate, ns = not significant.

2024 © healthdocbox.com Privacy Policy | Terms of Service | Feedback