All you need for your T cell research

Transcription

1 All you need for your T cell research Gentle sample preparation Accurate cell separation Optimized cell culture Precise flow cytometry

2 Products for isolation of mouse T cells T cells CD3ε MicroBead Kit # CD90.1 MicroBeads # CD90.2 MicroBeads # CD5 (Ly-1) MicroBeads # Pan T Cell II # CD4 + T cells CD4 (L3T4) MicroBeads # CD4 + T Cell # Treg cells CD4 + CD25 + Regulatory T Cell # CD25 MicroBead Kit # CD62L (L-selectin) MicroBeads # CD8 + T cells CD8a (Ly-2) MicroBeads # CD8a + T Cell # γ/δ + T cells TCRγ/δ + T Cell # T cell subsets Naive CD4 + T cells Naive CD4 + T Cell # CD4 + CD62L + T Cell # Memory T cells Pre-selection with: CD4 + or CD8 + T Cell # (CD4) # (CD8) CD62L (L selectin) MicroBeads # CD62L (L-selectin) MicroBeads # Naive CD8a + T cells Naive CD8a + T Cell # inkt cells NK1.1 + inkt Cell # Th1 cells Mouse IFN-γ Secretion Assay Cell Enrichment and Detection Kit (PE) # Th17 cells Mouse IL-17 Secretion Assay Cell Enrichment and Detection Kit (PE) # Th2 cells Mouse IL-4 Secretion Assay Cell Enrichment and Detection Kit (PE) # Activated T cells CD25 MicroBead Kit # CD154 Enrichment and Detection Kit (PE) # For rat and non-human primate cell isolation products, please visit: miltenyibiotec.com/cellseparation 2

3 Products for isolation of human T cells T cells and subsets T cells DN T cells CD3 MicroBeads # Whole Blood CD3 MicroBeads # CD2 MicroBeads # MACSxpress Pan T Cell # Pan T Cell # CD6 MicroBeads # CD28 MicroBead Kit # Double-negative T Cell # Naive Pan T cells Naive Pan T Cell # CD4 + T cells CD4 MicroBeads # CD4 MultiSort Kit # Whole Blood CD4 MicroBeads # CD4 + T Cell # MACSxpress CD4 T Cell # Treg cells CD25 MicroBeads II # CD127 MicroBead Kit # CD4 + CD25 + Regulatory T Cell # CD4 + CD25 + CD127 dim/ Regulatory T Cell II # CD25 + CD49d Regulatory T Cell # CD4 + CD25 + CD45RA + Regulatory T Cell # MACSxpress Treg # γ/δ + T cells Anti-TCRγ/δ MicroBead Kit # TCRγ/δ + T Cell # CD8 + T cells CD8 MicroBeads # CD8 MultiSort Kit # Whole Blood CD8 MicroBeads # CD8 + T Cell # MACSxpress CD8 T Cell # NKT cells CD3 + CD56 + NKT Cell # Anti-iNKT MicroBeads # Activated T cell subsets CD25 MicroBeads II # CD30 MicroBeads # CD69 MicroBead Kit II # CD154 MicroBead Kit # CD137 MicroBead Kit # Pre-selection of CD8 + T cells with: CD8 + T Cell # CD8 MultiSort Kit # Pre-selection of CD4 + T cells with: CD4 + T Cell # CD4 MultiSort Kit #

4 CD4 + T cell subsets CD8 + T cell subsets CD4 + RTE CD4 + Recent Thymic Emigrant # Naive CD4 + T cells Naive CD4 + T Cell II # Pre-selection with: CD4 + T Cell # CD62L MicroBeads # CD45RA MicroBeads # CD45RO MicroBeads # Skin-homing T cells CD4 + CLA + T Cell # Anti-CLA MicroBead Kit # Naive CD8 + T cells Naive CD8 + T Cell # Pre-selection with: CD8 + T Cell # CD45RA MicroBeads # CD45RO MicroBeads # Memory CD8 + T cells CD8 + Memory T Cell # Memory CD4 + T cells Memory CD4 + T Cell # MACSxpress CD4 Memory T Cell # Tem cells CD4 + Effector Memory T Cell # Tcm cells CD4 + Central Memory T Cell # Temra cells CD8 + CD45RA + Effector T Cell # Cytotoxic T cells CD8 + CD57 + T Cell # Th1 cells IFN-γ Secretion Assay Cell Enrichment and Detection Kit (PE) # Th2 cells CD294 (CRTH2) MicroBead Kit # IL-4 Secretion Assay Cell Enrichment and Detection Kit (PE) # Th17 cells IL-17 Secretion Assay Cell Enrichment and Detection Kit (PE) # Effector T cells CD27 MicroBeads # Color code for the use of MACS Cell Separation products Legend Positive isolation (labeling of target cells) Depletion (labeling for removal of an unwanted cell type) Isolation of untouched cells using a depletion cocktail Positive isolation of cells directly from whole blood Sequential separation (combination of depletion of non-target cells and positive selection) Isolation of untouched cells directly from whole blood inkt: NKT: TCM: Tem: Temra: Th: Treg: Invariant natural killer T cell Natural killer T cell Central memory T cell Effector memory T cell Effector memory CD45RA + T cell T helper cell regulatory T cell 4

5 Table of contents gentlemacs Dissociators gentlemacs Disscociators gentlemacs Tubes MACS Tissue Dissociation Kits MACS SmartStrainers SAMPLE PREPARATION 6 MACS MultiStand with MACS Columns and Separators MACS MicroBeads and Cell s MACS Whole Blood MicroBeads and MACSxpress Kits MACS Cytokine Secretion Assays Cell Enrichment and Detection Kits automacs Pro and MultiMACS Cell24 Separators CELL SEPARATION 8 TexMACS Medium MACS Cytokines and functional-grade antibodies Polyclonal T cell stimulation Antigen-specific T cell stimulation TexMACS Medium for T cells CELL CULTURE 14 MACSQuant Analyzer 10 Vio Dyes Recombinant antibodies for flow cytometry REAfinity Antibodies MACSPlex and Rapid Cytokine Inspector MACSQuant Flow Cytometers FLOW CYTOMETRY 18 5

6 SAMPLE PREPARATION Start smart The secret to success lies in your starting material gentlemacs Dissociators Versatile benchtop tissue dissociators that can process up to eight samples in parallel. Programmable and with optional temperature regulation to ensure standardized tissue dissociation. Gentle: Get viable single-cell suspensions with preserved epitopes Reproducible: Pre-defined programs for standardized tissue processing Fast: Process up to eight samples in parallel with the gentlemacs Octo Dissociator Safe: Sample processing in a closed and sterile system gentlemacs Tubes Tubes for the gentlemacs Dissociators, precision engineered to guarantee highest quality tissue dissociation or homogenization. gentlemacs C Tube: Use the purple C Tube for high yields of viable single cells gentlemacs M Tube: Choose the orange M Tube to thoroughly homogenize cells for, e.g., molecular analyses GET A DEMO Figure 2: gentlemacs C and M Tubes provide efficient dissociation or homogenization of any tissue sample. Figure 1: The gentlemacs Instruments versatile and reliable benchtop dissociators. GET A DEMO To arrange for an instrument demo in your laboratory, please contact your local sales representative or customer service. 6

7 SAMPLE PREPARATION Enzymatic tissue dissociation MACS Tissue Dissociation Kits Combine the gentlemacs Instrument s mechanical dissociation with enzymatic treatment for excellent yield, high viability, and preserved cell epitopes. Reproducible T cell isolation even from hard-to-process human and mouse tissues, including: Lamina propria (fig. 4) Tumor Lung Liver Spleen Various dissociation kits and programs as well as comprehensive technical support are also available for many other tissue types. VIDEO Not just cell strainers: SmartStrainers Easily remove aggregates and debris for optimal cell labeling and culture conditions. CD3-PE 10² 10¹ 10⁰ See the SmartStrainers in action! miltenyibiotec.com/smartstrainers CD8a-PE 10² 10¹ 10⁰ 10 ¹ 10 ¹ 10 ² 10 ² 10 ¹ 10⁰ 10¹ 10² 10 ² 10 ² 10 ¹ 10⁰ 10¹ 10² CD49b-APC CD4-APC Figure 4: Dissociation of mouse lamina propria tissue using the gentlemacs Dissociator in combination with the Lamina Propria Dissociation Kit, mouse. Subsequently cells were stained with CD3-PE, CD49b-APC, CD8a-PE, and CD4-APC antibodies and analyzed using the MACSQuant Analyzer. Figure 3: MACS Tissue Dissociation Kits specifically designed for optimal dissociation of the respective tissue. For a complete list of our sample preparation products, please visit: miltenyibiotec.com/samplepreparation 7

8 CELL SEPARATION Select the best Choose the optimal solution for your experiment MACS Technology One portfolio for all your T cell isolation needs: With the MACS Cell Separation portfolio, you enjoy the freedom to choose the cell isolation option that s best for your specific requirements. Our proven magnetic cell separation technology is continuously expanding to offer new and innovative options across basic and clinical research. Unsurpassed possibilities for the isolation of T cells: Nano-sized beads (column-based technology with MACS MicroBeads, Cell s, and Whole Blood MicroBeads) for highest purity and recovery Micro-sized beads (column-free MACSxpress Technology) for maximum convenience and fastest isolations Nano-sized beads Micro-sized beads Benefits The most comprehensive cell isolation technology available Applicable to virtually any species or sample material Limitless range of use from basic to clinical research Cell isolation from whole blood within 20 minutes No density gradient centrifugation High reproducibility due to minimal sample handling Components MACS Columns MACS Separators MACS MicroBeads MACSxpress Separator MACSxpress Beads Starting material PBMCs Whole blood Bone marrow Single-cell suspensions Whole blood MACS MicroBead Technology MACSxpress Technology For a complete list of our cell separation products, please look inside the front cover or visit: miltenyibiotec.com/cellseparation 8

9 CELL SEPARATION Isolate virtually any T cell subset MACS MicroBeads Straightforward positive selection of your T cell subset of choice via the respective markers. Highest purity and recovery as simple as it gets (fig. 5). Isolation of any rare or frequently occurring T cell type Minimal cell labeling without cell activation From small- to large-scale separations Proven technology for basic research and clinical applications CD8-PE CD8-PE Before separation CD56-FITC CD56-FITC CD8-PE CD8-PE After separation CD56-FITC CD56-FITC MACS Cell s Remove any unwanted cells from the target population by depletion, when untouched cell separation or subsequent magnetic sorting steps are required (fig. 6). Untouched isolation of almost every T cell subset Fully compatible with any downstream application Ideal for pre-enrichments (e.g. CD4 + cell isolation prior to flow sorting of regulatory T (Treg) cells) Discover diversity Isolate T cell subsets from human, mouse, rat, and nonhuman primate samples. Choose from Miltenyi Biotec s huge portfolio for the isolation of virtually any T cell subset, including: Naive T cells (pan, CD4 +, CD8 + ) Memory T cells (CD4 +, CD8 +, Tem, Tcm) Th1, Th2, Th17 cells Treg cells γ/δ T cells NKT cells and many more Figure 5: Enrichment of CD8 + T cells from human PBMCs using CD8 MicroBeads, human. Cells were stained with CD8-PE and CD56-FITC and analyzed by flow cytometry on the MACSQuant Analyzer. CD3ε-PE 10² 10¹ Before separation 10¹ 10² CD4-APC ¹ 10² Figure 6: Untouched isolation of CD4 + T Cells from a mouse spleen using the CD4 + T Cell, mouse. The spleen single-cell suspension was prepared on the gentlemacs Dissociator. Cells were stained with CD4-APC and CD3ε-PE antibodies and analyzed on the MACSQuant Analyzer. 10² 10¹ After separation FAST PROTOCOL Isolate your T cells faster than ever 18-minute protocols are available for your favorite Cell s: CD3ε-PE miltenyibiotec.com/fastprotocol CD4-APC 9

10 CELL SEPARATION Isolate T cells directly from whole blood MACS Whole Blood MicroBeads Positive selection of pan T cells, CD4 + or CD8 + cell subsets from human whole blood or bone marrow samples (fig. 8). Effortless, simple, and fully automated with the automacs Pro Separator. Excellent purity and recovery No density gradient centrifugation or erythrocyte lysis Fully automatable for easy standardization Less hands-on time higher safety CD14-APC 10² 10¹ Before separation 10¹ 10² CD3-PE CD14-APC 10² 10¹ After separation 10¹ 10² CD3-PE VIDEO Automated cell separation: automacs Pro Separator Walk-away convenience for your favorite MACS MicroBead Kits, s, and Whole Blood MicroBeads. Cell separation from whole blood using the automacs Pro Separator miltenyibiotec.com/videoautomacspro Figure 8: Enrichment of CD3 + T cells directly from human whole blood using Whole Blood CD3 MicroBeads and the MultiMACS Cell24 Separator. Cells were stained with CD3-PE and CD14-APC and analyzed on the MACSQuant Analyzer. Cells were triggered via CD45-VioBlue, cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide staining. Magnetic labeling Add the MACSxpress Cell Isolation Cocktail to up to 30 ml whole blood and incubate on an overhead rotator (e.g. MACSmix Tube Rotator). MACSxpress Kits Fast and easy untouched isolation of pan T cells and several CD4 + and CD8 + cell subsets via depletion of unwanted cells directly from human whole blood. Go from whole blood to pure cells within 20 minutes No density gradient or erythrocyte lysis required For up to 30 ml of whole blood Column-free for maximum convenience Magnetic separation Place open tube in the MACSxpress Separator. 5 min 15 min Collection of target cells Collect the supernatant which contains the unlabeled target cells. Total: 20 min Figure 7: The MACSxpress Separator perfectly suited to process large volumes of whole blood. Figure 9: Fast and easy cell separation from whole blood using MACSxpress Technology. 10

11 CELL SEPARATION Isolate viable cytokine-secreting T cells MACS Cytokine Secretion Assays Cell Enrichment and Detection Kits Available only from Miltenyi Biotec: detection and enrichment of viable cytokine-secreting T cell subsets. Unique: Isolation of viable cytokine-secreting T cells Accurate: Analysis of cytokine secretion at single-cell level Sensitive: Detection of one T cell in a million cells Versatile: Isolate human and mouse T cell subsets such as Th1 (IFN-γ), Th2 (IL-4), Th17 (IL-17), and IL-10-secreting Tr1 cells Cytokine Catch Reagent Stimulated leukocyte The cells are stimulated in vitro. T cells or other stimulated leukocytes rapidly start to secrete cytokines hours A cytokine-specific affinity matrix, the Cytokine Catch Reagent, is attached to the cell surface of all leukocytes via the CD45 antigen. You can use isolated cells right away for downstream applications such as: Generation of influenza-specific cytotoxic T cells (human IFN-γ assay) Functional analysis of the Th1 and Th17 subsets (human IFN-γ and IL-17 assays) Characterization of allergen-specific Tr1 cells (human IL-10 assay) Studies on memory T cell subsets (human IL-10 assay) Studies on transplantation tolerance with isolated Tr1 cells (mouse IL-10 assay) Cytokinesecreting cell Secreted cytokines PE-conjugated Cytokine Detection Antibody 5 min During a short incubation period at 37 C the secreted cytokines bind to the Cytokine Catch Reagent of the respective cells. 45 min The cells are stained with, e.g., a specific PE-conjugated Cytokine Detection Antibody and are ready for analysis by flow cytometry. 10 min Anti-PE MicroBead Optional: If magnetic enrichment is required, the cells can be magnetically labeled with Anti-PE MicroBeads. 15 min The cells can be efficiently magnetically enriched using MACS Columns and MACS Separators: Cells remain viable for downstream applications such as expansion and functional characterization. Magnetic enrichment of cytokine-secreting cells enables unmatched sensitivity of detection as low as one cell in a million. 15 min Figure 10: Principle of the Cytokine Secretion Assays. 11

12 CELL SEPARATION Automated cell separation automacs Pro Separator Fully automated benchtop separator for magnetic cell sorting of multiple samples. Automated, true walk-away cell labeling and isolation for up to six samples Standardized cell separation for reproducible, userindependent results Chilled sample processing (4 C) Fast and gentle isolation of virtually any cell type MultiMACS Cell24 Separator Plus Efficient, semi-automatic cell isolation from up to 24 samples simultaneously with complete ease. Time-saving: Up to 24 separations in parallel User-friendly: Easy to operate with intuitive touchscreen interface Adaptable: Use with single columns or column blocks GET A DEMO GET A DEMO Figure 11: The automacs Pro Separator. Fully automated cell labeling and cell separation on your benchtop. Figure 12: Parallel cell isolation from up to 24 samples with the MultiMACS Cell24 Separator Plus. VIDEO Simplify your workflow: MultiMACS Cell24 Separator Plus Easy, high-throughput cell isolation for your laboratory needs. miltenyibiotec.com/multimacs 12

13 CELL SEPARATION MACS Separators MACS Columns and Separators With its MACS Column and Separator portfolio, Miltenyi Biotec provides the perfectly tailored solution for your experimental needs. Explore the options below to learn more about our separation hardware. Manual MACS Cell Separation provides a simple and straightforward experimental setup for excellent separation results Automated multisample cell separation with the MultiMACS Cell24 and automacs Pro Separators. Flexible workflow solutions for higher throughput and best reproducibility MiniMACS Separator OctoMACS Separator MidiMACS Separator QuadroMACS Separator MACSxpress Separator automacs Pro Separator MultiMACS Cell24 Separator Columns Capacity, number of total cells Capacity, number of labeled cells MS Columns, Large Cell Columns MS: Large Cell: MS: Large Cell: *with Single-Column Adapter MS Columns, Large Cell Columns MS: Large Cell: MS: Large Cell: LS Columns, LD Columns, Whole Blood Columns LS: LD: Whole blood: up to 15 ml blood LS Columns, LD Columns, Whole Blood Columns LS: LD: Whole blood: up to 15 ml blood n/a Up to 30 ml whole blood automacs Columns cells or 15 ml whole blood Multi-24 Column Block, LS Columns*, LD Columns*, Whole Blood Columns* Multi-24 Column Block: LS: LS: LD: Whole blood columns: 10 ml whole blood LS: LD: LS: LD: n/a cells LS: LD: Multi-24 Column Block: Table 1: MACS Separators and Columns. 13

14 CELL CULTURE Culture is key Best conditions for your T cells MACS Cytokines MACS Cytokines are designed for demanding applications in cell culture, differentiation studies, and functional assays in all areas of T cell research. Save time and costs, get consistent results. Superior quality: Available in research, premium, and GMP grade Reliable: Standardized, high biological activity Flexible: Small, bulk, and customized fillings and cytokine cocktails Stimulation reagents For your flexibility Miltenyi Biotec offers a huge variety of unique solutions for activation and expansion of human and mouse T cells, with: Antigen-specific stimulation Polyclonal stimulation Functional-grade antibodies Mimic or inhibit ligand-receptor interactions for the activation and expansion of human and mouse T cells. Get safe and reliable results from best-quality antibodies. Low endotoxin content Free of preservatives Sterile filtered Pure (e.g. CD3 pure, CD28 pure) or biotinylated Up to GMP grade TexMACS Media Optimized cell culture media developed for highperformance cultivation of human and mouse T cells. Get optimal cell growth, high expansion rates, and reliable activation even without serum. Optimized for T cell and Treg cell cultures Animal component-free Figure 13: The specialized TexMACS Media and optimized stimulation reagents provide best T cell culture conditions. Figure 14: High-quality cytokines, with reliable lot-to-lot consistency and standardized, high biological activity ensure consistent cell culture results. For a complete list of Miltenyi Biotec s cell culture and stimulation reagents, please visit: miltenyibiotec.com/cellculture 14

15 CELL CULTURE Cytokines and functional antibodies T cell differentiation Miltenyi Biotec offers a large portfolio of T cell relevant cytokines, growth factors, and functional-grade antibodies to provide the best tools for your T cell differentiation experiments. One provider for all your needs benefit from our seamless assortment. Legend available from Miltenyi Biotec *available also as GMP grade activating antibody blocking or neutralizing antibody CD4 + naive T cell IL-2* anti CD3* anti CD28* IL-12 IL-18 IL-27 IFN-γ anti IL-4 IL-4* IL-6* IL-31 IL-23 TSLP IL-25 anti IFN-γ TGF-β1 IL-1β* IL-6* IL-21* IL-23 anti IL-2 anti IL-4 anti IFN-γ TGF-β1 TGF-β1 IL-4* anti IFN-γ IL-6* IL-21* CXCL13 IL-6* TNF-α* Th1 cell Th2 cell Th17 cell Treg cell Th9 cell Tfh cell Th22 cell Figure 15: Differentiation and activation of human T cells. For mouse and rat products, please visit miltenyibiotec.com/cytokines FOR SPECIALISTS CytoBox All-in-one kit, with everything you need for efficient Th1, Th2, and Th17 differentiation of mouse naive CD4 + T cells including cytokines and functional-grade antibodies. miltenyibiotec.com/cytokines 15

16 CELL CULTURE Antigen-specific T cell stimulation PepTivator Peptide Pools Extensive panel of virus-, tumor-, and fungus-specific antigen peptides (e.g. cytomegalovirus (CMV), MAGE, A. fumigatus) for the effective stimulation of antigenspecific CD4 + and CD8 + T cells (fig. 16). Efficient: 15-mer peptides with 11-amino-acid overlaps, covering the complete sequence of the respective antigen Convenient: Easy reconstitution in water (no DMSO needed) Flexible: PepTivator Peptide Pools HT 96-well format peptide pools for high-throughput applications High-quality: Available in research, premium, and GMP grade Recombinant and purified proteins Stimulation of antigen-specific CD4 + and CD8 + T cells via whole proteins. Benefit from: Specifically designed, recombinant proteins for powerful T cell stimulation (e.g. CMV pp65) Purified lysates for antigen-specific CD4 + and CD8 + T cells (e.g. A. fumigatus, fig. 17) Available up to GMP grade Before separation Unstimulated After separation Unstimulated Stimulated CD4-FITC 10² 10¹ CD4-FITC 10² 10¹ CD4-APC CD4-APC CD4-APC ¹ 10² CD154-PE ¹ 10² CD154-PE Stimulated Anti-IFN-γ-PE Anti-IFN-γ-PE Anti-IFN-γ-PE Anti-IFN-γ-PE Before separation After separation CD8-FITC CD8-FITC ¹ 10² Anti-IFN-γ-PE Anti-IFN-γ-PE Anti-IFN-γ-PE Anti-IFN-γ-PE CD154-PE CD4-FITC 10² 10¹ CD4-FITC 10² 10¹ ¹ 10² CD154-PE Figure 16: Restimulation of human PBMCs with and without PepTivator CMV pp65 premium grade. For flow cytometry analysis cells were subsequently stained with Anti-IFN-γ-PE (intracellular cytokine staining) and counterstained for CD4 and CD8 expression. Figure 17: Stimulation of human PBMCs with A. fumigatus lysate. After stimulation, A. fumigatus specific T cells were stained and magnetically enriched according to their expression of CD154. Cells were subsequently stained with CD4-FITC, and CD45-VioBlue. Viable CD45 + lymphocytes are shown. 16

17 CELL CULTURE Polyclonal T cell stimulation T Cell Activation/Expansion Kits MACSiBead Particles loaded with functional antibodies to mimic cell-mediated T cell stimulation. The particles resemble artificial antigen-presenting cells. Highly efficient and homogeneous polyclonal T cell stimulation for optimal expansion (fig. 18) Specific kits for human and mouse T cells and Treg cells Flexible labeling with antibodies of your choice Cell number 10⁸ 10⁷ 10⁶ CD4 + T cells CD8 + T cells TransAct T Cell Reagent Polymeric nanomatrix conjugated to humanized CD3 and CD28 agonists. The next generation of T cell activation: easy, efficient, and translational. Ready to use Simple volumetric dosage No bead removal required: easy elimination by, e.g., culture wash Suitable for automated culture systems, e.g., CliniMACS Prodigy Up to GMP grade 10⁵ Time in culture (d) Figure 18: CD4 + and CD8 + T cell expansion rates achieved with the T Cell Activation/Expansion Kit, mouse. TransAct T Cell Reagent or T Cell Activation/Expansion Kits? Yes Optimized for human T cells Human T cells? No Mouse and human products available CytoStim Reagent Rapid and effective restimulation of human and non-human primate effector/memory T cells by cross-linking TCRs to MHC molecules. Save time and maximize your potential. Rapid cytokine expression and up-regulation of activation markers by target cells Antibody-based, non-toxic alternative to SEB Suitable for CD4 + and CD8 + T cells in fresh PBMCs, whole blood, or other single-cell suspensions Ready-to-use reagent desired? Cell number known? Simple volumetric dosage Reagent/bead removal desired? Easy removal by washing Yes No Yes No Yes No TransAct T Cell Reagent Separate particle loading step Dosage via bead:cell ratio Optional bead removal step T Cell Activation/Expansion Kits Figure 19: Decision tree how to stimulate your T cells. 17

18 FLOW CYTOMETRY Join the flow revolution Flow cytometry solutions to power your research Vio Dyes An exclusive family of fluorochromes for flow cytometry and fluorescence microscopy. Bright: Superior mean fluorescence intensity for excellent signals (fig. 20) Distinct: High stain index for clear population discrimination Precise: Ideal for multicolor experiments due to low spillover and marginal compensation MACS Antibodies Profit from fluorochrome-conjugated antibodies specifically designed for flow cytometry analysis of any immune cell. Powerful: Various bright dyes enhance target cell detection Versatile: Broad panel of human, mouse, rat, and non-human primate antibodies Customizable: Personalized solutions for conjugates and multicolor cocktails FITC conjugates become as bright as PE conjugates Figure 20: VioBright FITC equals PE in fluorescence intensity and surpasses standard FITC conjugates. Human CD4 + T cells were stained with CD4-PE, CD4-VioBright FITC, and CD4-FITC antibodies and subsequently analyzed by flow cytometry. A CD4-PE Mouse monoclonal Da CD4-FITC CD4-VioBright FITC Da REAfinity Antibodies Recombinantly engineered antibodies with unique advantages compared to conventional human, mouse, or rat monoclonal antibodies. High purity and lot-to-lot consistency for greater reproducibility (fig. 21) No tedious and costly FcR blocking One universal isotype control (IgG1) Over 500 specificities B Lower panel: From Rea Antibodies page: antibodies-and-dyes/recombinant-antibodies. REAfinity Antibody aspx Panel High Dapurity- no Ig, Fig ??? Da FOR SPECIALISTS MACS Marker Screen Characterization and profiling of human cell lines, primary cells, and cell mixtures. Easy and convenient marker profiling 371 monoclonal antibodies readily arrayed onto four 96-well plates Pre-titrated, and validated for flow analysis miltenyibiotec.com/markerscreen Figure 21: Superior purity of REAfinity Antibodies, demonstrated by mass spectrometry analysis of mouse monoclonal (A) and REAfinity Antibody samples (B). The standard mouse monoclonal antibody shows a mixture of antibody chains, while the REA clone only shows one heavy chain (50 kda) and one light chain (25 kda). 18

19 FLOW CYTOMETRY Cytokine analysis Rapid Cytokine Inspector High-throughput multiparameter analysis of activated human T cells. Easy, convenient, and fast. Rapid flow cytometric detection of cytokine production in activated CD4 + and CD8 + T cells Detection of surface markers and intracellular cytokines in one experiment Suited for human whole blood and PBMC samples Select an antibody against your cytokine of interest: Anti-IFN-γ (PE, APC), Anti-TNF-α (APC), Anti-IL-2 (PE), Anti-IL-17 (PE, APC) MACSPlex Cytokine Kits Determine multiple soluble cytokines in a single human or mouse T cell sample using standard flow cytometry techniques. No need for expensive extra instruments. True multiplexing Up to 12 human or 10 mouse cytokines in one sample Optimized for automated measurement using the Express Mode of the MACSQuant Analyzers Fluorochrome CD4/CD8 T Cell Detection Cocktail CD4 T Cell Detection Cocktail VioBlue CD3 CD3 CD3 FITC CD8 CD154 CD8 CD8 T Cell Detection Cocktail PE Cytokine 1 Cytokine 1 Cytokine 1 PerCP CD14/CD20 CD14/CD20 CD14/CD20 APC CD4 CD4 Cytokine 2 FcR-Block FcR-Block FcR-Block Table 2: Rapid Cytokine Inspector Kits are designed for intracellular cytokine detection and contain everything you need for staining cytokineproducing T cells. Choose the appropriate anti-cytokine antibody (e.g. Anti-IFN-γ, Anti-TNF-α, Anti-IL-2, Anti-IL-17) and start your experiment. MPx Capture Beads coupled to anti-cytokine antibodies Cytokines bind to specific antibodies on MPx Capture Beads FOR SPECIALISTS Rapid ARTE Rapid Antigen-Reactive T Cell Enrichment High-resolution characterization of rare CD154 + antigen-specific human T cells Unique combination of magnetic enrichment and flow analysis Only 60 minutes processing time Low background high statistical significance Cytokines bound to specific MPx Capture Beads are labeled with APC-conjugated anti-cytokine antibodies Check out our special protocol at miltenyibiotec.com/protocols MACSPlex Bead (MPx) Cytokines Detection Reagent Figure 22: Principle of MACSPlex analysis. For a complete list of Miltenyi Biotec s flow cytometry reagents and instruments, please visit: miltenyibiotec.com/flowcytometry 19

20 FLOW CYTOMETRY The MACSQuant Instruments MACSQuant Flow Cytometers Fully automated and powerful benchtop flow cytometers for highly sensitive, multiparameter cell analyses with walk-away convenience. MACSQuant Analyzer 10: Three lasers (violet, blue, red) and ten optical parameters MACSQuant VYB: Unique variant with a yellow laser for optimized detection of fruit fluorescent proteins Volumetric cell counting without reference beads Robotic processing of up to 96 samples Automated labeling, pre-enrichment, compensation, cleaning, and shutdown MACSQuant Cell Sorter The MACSQuant Tyto revolutionizes cell sorting with high speed, ten parameters, and unparalleled operator safety and cell viability. Three lasers for up to ten optical parameters Fully enclosed, disposable cartridges for sample sterility and operator safety Never before-seen cell viability due to the gentle microchip-based sorting process Engineered for ease of use fully automated, waste bin-free, and nozzle-free sorting GET A DEMO GET A DEMO Figure 23: MACSQuant Flow Cytometers sensitive, accurate, and powerful benchtop instruments with unique automation features for hands-free multisample processing. Figure 24: The MACSQuant Tyto revolutionizes cell sorting. High speed, 10-parameter sorting with unrivaled cell viability. Powerful flow: MACSQuant Instruments Automate your flow cytometry analyses miltenyibiotec.com/colorflow VIDEO 20

21 A complete translational T cell workflow Research Clinical MACS Sample Preparation gentlemacs Instruments gentlemacs Tubes MACS Tissue Dissociation Kits Preparation MACS Cell Separation CD8 MicroBeads CD4 + T Cell Additional subsets including CD45RO, CD62L, and regulatory T cells Separation CliniMACS CD8 Reagent CliniMACS CD4 Reagent CliniMACS CD62L Reagent CliniMACS CD25 Reagent CliniMACS System MACS Cell Culture TexMACS Medium T Cell Activation and Expansion Kits Cytokines Activation TexMACS GMP Medium* MACS GMP CD3 pure* MACS GMP CD28 pure* MACS GMP ExpAct Treg Kit* MACS GMP T Cell TransAct* MACS GMP Products* MACSmolecular MACSductin Reagent for cell transduction MACSelect System for selecting transfected cells Gene modification and monitoring MACSQuant VYB with violet, yellow, and blue laser (RUO) Simultaneous analysis of fluorescent proteins such as GFP, YFP, mcherry, dsred, and others (RUO) MACS Flow Cytometry (RUO) MACS Cell Culture TexMACS Medium MACS Cytokines including IL-2, IL-7, IL-15, and IL-21 Expansion TexMACS GMP Medium* MACS GMP-grade Cytokines* including IL-2, IL-7, IL-15, and IL-21 MACS GMP Cell Culture Bags* MACS GMP Products* MACS Flow Cytometry (RUO) MACSQuant Flow Cytometers, Express Mode Rapid Cytokine Inspector Kit Kits for functional testing Downstream analysis Cryopreservation CryoMACS Freezing Bags CryoMACS DMSO 10 (RUO, EP, USP) CryoMACS Freezing Products RUO=Research Use Only EP=European Pharmacopoeia USP=United States Pharmacopeia *MACS GMP Products are for research use and ex vivo cell culture processing only, and are not intended for human in vivo applications. For complete regulatory and legal notices please refer to page 22 (back cover). 21

22 miltenyibiotec.com/tcells Germany/Austria/ Switzerland Miltenyi Biotec GmbH Friedrich-Ebert-Straße Bergisch Gladbach Germany Phone Fax USA/Canada Miltenyi Biotec Inc Lindbergh Street Auburn, CA 95602, USA Phone 800 FOR MACS Phone Fax Australia Miltenyi Biotec Australia Pty. Ltd. Unit 16 A, 2 Eden Park Drive Macquarie Park NSW 2113 Australia Phone Fax macs@miltenyibiotec.com.au Benelux Miltenyi Biotec B.V. Schipholweg 68 H 2316 XE Leiden The Netherlands macs@miltenyibiotec.nl Customer service The Netherlands Phone Fax Customer service Belgium Phone Fax Customer service Luxembourg Phone Fax China Miltenyi Biotec Technology & Trading (Shanghai) Co., Ltd. Room 2309 No. 319, Xianxia Road Changning District Shanghai, P.R. China Phone Fax macs@miltenyibiotec.com.cn France Miltenyi Biotec SAS 10 rue Mercoeur Paris, France Phone Fax macs@miltenyibiotec.fr Italy Miltenyi Biotec S.r.l. Via Persicetana, 2/D Calderara di Reno (BO) Italy Phone Fax macs@miltenyibiotec.it Japan Miltenyi Biotec K.K. Nittsu-Eitai Building 5F Fuyuki, Koto-ku, Tokyo , Japan Phone Fax macs@miltenyibiotec.jp Nordics and Baltics Miltenyi Biotec Norden AB Scheelevägen Lund Sweden macs@miltenyibiotec.se Customer service Sweden Phone Fax Customer service Denmark Phone Fax Customer service Norway, Finland, Iceland, and Baltic countries Phone Fax Singapore Miltenyi Biotec Asia Pacific Pte Ltd. 100 Beach Road #28-06 to Shaw Tower Singapore Phone Fax macs@miltenyibiotec.com.sg Miltenyi Biotec provides products and services worldwide. Visit to find your nearest Miltenyi Biotec contact. South Korea Miltenyi Biotec Korea Co., Ltd Arigi Bldg. 8F 562 Nonhyeon-ro Gangnam-gu Seoul 06136, South Korea Phone Fax macs@miltenyibiotec.co.kr Spain Miltenyi Biotec S.L. C/Luis Buñuel 2 Ciudad de la Imagen Pozuelo de Alarcón (Madrid) Spain Phone Fax macs@miltenyibiotec.es United Kingdom Miltenyi Biotec Ltd. Almac House, Church Lane Bisley, Surrey GU24 9DR, UK Phone Fax macs@miltenyibiotec.co.uk Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. MACS GMP Products are for research use and ex vivo cell culture processing only, and are not intended for human in vivo applications. For regulatory status in the USA, please contact your local representative. MACS GMP Products are manufactured and tested under a quality system certified to ISO and are in compliance with relevant GMP guidelines. They are designed following the recommendations of USP <1043> on ancillary materials. The CliniMACS System components, including Reagents, Tubing Sets, Instruments, and PBS/EDTA Buffer, are designed, manufactured and tested under a quality system certified to ISO In the EU, the CliniMACS System components are available as CE-marked medical devices for their respective intended use, unless otherwise stated. The CliniMACS Reagents and Biotin Conjugates are intended for in vitro use only and are not designated for therapeutic use or direct infusion into patients. The CliniMACS Reagents in combination with the CliniMACS System are intended to separate human cells. Miltenyi Biotec as the manufacturer of the CliniMACS System does not give any recommendations regarding the use of separated cells for therapeutic purposes and does not make any claims regarding a clinical benefit. For the manufacturing and use of target cells in humans the national legislation and regulations e.g. for the EU the Directive 2004/23/EC ( human tissues and cells ), or the Directive 2002/98/EC ( human blood and blood components ) must be followed. Thus, any clinical application of the target cells is exclusively within the responsibility of the user of a CliniMACS System. In the US, the CliniMACS CD34 Reagent System, including the CliniMACS Plus Instrument, CliniMACS CD34 Reagent, CliniMACS Tubing Sets TS and LS, and the CliniMACS PBS/EDTA Buffer, is FDA approved; all other products of the CliniMACS Product Line are available for use only under an approved Investigational New Drug (IND) application or Investigational Device Exemption (IDE). CliniMACS MicroBeads are for research use only and not for human therapeutic or diagnostic use. CryoMACS Freezing Bags are manufactured by Miltenyi Biotec GmbH and controlled under an ISO13485 certified quality system. These products are available in Europe as CE-marked medical devices and are marketed in the USA under FDA 510(k) clearance. CliniMACS, CliniMACS Prodigy, CryoMACS, CytoStim, automacs, ExpAct, gentlemacs, MACS, MACSductin, MACSmix, MACSQuant, MACSelect, MACSxpress, MidiMACS, MiniMACS, MultiMACS, OctoMACS, PepTivator, QuadroMACS, REAfinity, TexMACS, TransAct, Tyto, Vio, VioBlue, VioBright, and the MACS logo are registered trademarks or trademarks of Miltenyi Biotec GmbH and/or its affiliates in various countries worldwide. Copyright 2016 Miltenyi Biotec GmbH and/or its affiliates. All rights reserved