Flow Cytometry Support Reagents

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1 Excite and inspire Flow Cytometry Support Reagents Introduction Miltenyi Biotec is a leading supplier of flow cytometry products, offering one of the broadest ranges of antibodies, kits, assays, and support reagents available. This includes the Vio Dye range of fluorescently conjugated antibodies, incorporating conjugates such as VioBlue, VioGreen TM, PerCP-Vio700, PE-Vio770 TM, and APC-Vio770, allowing Miltenyi Biotec to further expand standard panels with these stable and robust dyes. In addition to the Vio Dye range, Miltenyi Biotec also offer a complete set of flow cytometry support reagents, which include instrument support reagents, sample preparation reagents, fluorescence enhancement reagents, and a broad range of viability, conjugation, and staining kits. Table of contents MACSQuant Instrument support reagents MACS Compensation Reagents MACSQuant Calibration Beads MACSQuant Buffers Sample preparation reagents Red Blood Cell Lysis Solution (10 ) Inside Stain Kit Fluorescence Enhancement reagents FcR Blocking Reagents FASER Kits Viability, conjugation, and staining kits Annexin V Reagents and Kits Propidium Iodide Solution Live Cell Discrimination Kit Fixation and Dead Cell Discrimination Kit MACSQuant Instrument support reagents MACS Compensation Reagents The MACS Compensation Reagents are a dual antibody system that allows for the compensation of any given PE- or APC-conjugated antibody and several tandem fluorochrome conjugates. In contrast to bead-based compensation systems, the cell-based MACS Compensation Reagents system takes into account the autofluorescence of cells during compensation and avoids overcompensation. Equal quantities of stained and unstained cells are mixed and compensation is performed using a lymphocyte gate. The MACS Compensation Reagents employ a dual antibody system that selectively targets CD45-expressing cells (all leukocytes) as well as the fluorochrome moiety of the antibody to be compensated for. Human cells are incubated with the MACS Compensation Reagents for labeling via CD45 after which the fluorochrome -conjugated antibody in question is added. Thus, all CD45 + cells within the sample are fluorescently labeled with the target antibody irrespective of the antibody s specificity. Product Capacity Order no. MACS Comp Reagent (APC), human MACS Comp Reagent (PE), human MACS Comp Reagent (APC), mouse MACS Comp Reagent (PE), mouse For For For For

2 MACSQuant Calibration Beads The MACSQuant Calibration Beads are designed for routine calibration of the MACSQuant Flow Cytometers. These calibration particles are 2 and 3 μm in size and enable the adjustment of the voltage settings. In addition, the 3 μm size particles are stained with different fluorescent dyes, whose emission spectra are compatible with many commonly used fluorochromes for flow cytometry. The MACSQuant Calibration Beads are excited between 400 nm and 650 nm to yield emission in all fluorescent channels. This enables the successful calibration of the laser settings. Sample preparation reagents Red Blood Cell Lysis Solution (10 ) Red Blood Cell Lysis Solution (10 ) has been developed for the lysis of red blood cells to ensure optimal lysis of erythrocytes with minimal effect on leukocytes. A washing step after erythrocyte lysis is optional, depending on the application. The solution is suitable for lysis of erythrocytes in single-cell suspensions of human, mouse, or rat origin. Product Capacity Order no. MACSQuant Calibration Beads For A B MACSQuant Buffers The MACSQuant Buffers are ready-to-use solutions supplied in 1.5 L bottles that can be connected directly to the MACSQuant Flow Cytometers for optimal instruments operation. The MACSQuant Running Buffer has been developed for the optimal processing and efficient acquisition of cells. The MACSQuant Washing Solution has been developed for the routine cleaning and rinsing of the fluidics of the instruments. This solution contains detergents as well as stabilizer for an efficient cleaning of the system. The MACSQuant Storage Solution has been developed for overnight or long-term storage. The MACS Bleach Solution is used to safely decontaminate the instrument fluidics for periodic decontamination and maintenance. Product Content Order no. MACSQuant Starting Buffer Kit L MACSQuant Running Buffer L MACSQuant Washing Solution L MACSQuant Storage Solution L MACSQuant Washing and Storage Solution Kit L MACSQuant Washing Solution L MACSQuant Storage Soluton MACS Bleach Solution 1 L Side scatter Forward scatter Figure 1: Erythrocytes in human peripheral blood were lysed with Red Blood Cell Lysis Solution followed by a washing step (A), or without washing step (B) and analyzed by flow cytometry. Dead cells were excluded from the analysis based on propidium iodide fluorescence. Product Capacity Content Order no. Red Blood Cell Lysis Solution (10 ) For 50 ml of whole blood 50 ml Inside Stain Kit The Inside Stain Kit is a unique tool for intracellular staining of cells in suspension, cells on slides, or cells immobilized on a MACS Separation Column. For the latter protocol, target cells are magnetically enriched on an MS Column according to the expression of a chosen antigen. After elution, the cells are fixed with Inside Fix and directly applied onto a second column. Permeabilization, intracellular staining, and washing steps are performed whilst the cells are retained on the column. The stained cells are eluted and can subsequently be analysed by immunocytochemistry or flow cytometry. This approach is easy to perform, requires small volumes of staining reagents, and cell loss is minimal compared to conventional staining techniques. Thus, the Inside Stain Kit is an ideal tool, for example for the detection of enriched tumor cells. Typical applications for the Inside Stain Kit are: Immunocytochemical detection and analysis of enriched epithelial cells based on the expression of intracellular antigens, for example, cytokeratins. Analysis of clonality of enriched B cell lymphoma or myeloma cells by staining for intracellular immunoglobulin light chains. Intracellular cytokine staining in pre-enriched T cells, dendritic cells, etc. Side scatter Forward scatter Product Capacity Components Order no. Inside Stain Kit For ml Inside Fix ml Inside Perm

3 Positive selection of magnetically labeled cells using an MS Column. Elution of magnetically labeled cells and fixation by adding Inside Fix. Fluorescence enhancement reagents FcR Blocking Reagents Optimises staining and signal-to-noise ratio Inhibits non-specific Fc Receptor binding Incubation with FcR Blocking Reagents increase the specificity of antibody or MicroBead labeling and thereby improves the purity of target cells, including extremely rare target cells such as antigen-specific B cells, fetal cells in maternal blood, stem cells, or disseminated epithelial tumor cells. Relative cell number Relative cell number CD90-FITC CD90-FITC Application of the fixed cells to a second column. Permeabilization with Inside Perm. Application of staining reagents to the column. Figure 3: Mouse spleen cell suspensions were incubated with (A) and without (B) FcR Blocking Reagent, and subsequently stained with CD90-FITC (# ). Cells were analyzed by flow cytometry. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence. Product Content Capacity Order no. FcR Blocking Reagent, human 2 ml For FcR Blocking Reagent, mouse 1 ml For Elution, substrate incubation and flow cytometric or immunocytochemical evaluation. Figure 2: In-column intracellular staining of cells isolated with MACS Technology. FASER Kits Enlightening: brightens up dim fluorescence signals Clear-cut: accurately discriminates cell populations through increased sensitivity No-compromise: use the same epitope for cell sorting and analysis The FASER (Fluorescence Amplification by Sequential Employment of Reagents) Kits can be used to amplify weak fluorescence intensity resulting from staining of lowexpressed antigens, the use of low-affinity antibodies, or immunomagnetic and fluorochrome-conjugated antibody labeling of cells via the same epitope. Furthermore, increasing the fluorescence intensity simplifies the flow cytometric discrimination of labeled and non-labeled cell fractions. The FASER Kits can also be used in combination with Anti-FITC, Anti-PE, or Anti-APC MicroBeads for the enhancement of weak magnetic labeling. The FASER Kits FITC, PE, and APC were developed to increase the fluorescence intensity of cells labeled with virtually any antibody conjugated to FITC, PE or APC. Fluorescence intensity is amplified by the sequential addition of two reagents: The fluorochrome-specific Activator (Reagent 1) and the fluorochrome-conjugated Enhancer (Reagent 2). For further enhancement of

4 fluorescence intensity, the sequential addition of reagents can be performed as often as required. The FASER Kits do not affect direct staining with other fluorochromeconjugated antibodies or MACS MicroBeads but should not be used in combination with biotin-conjugated antibodies. They are suitable for fresh or formaldehyde-fixed cells in suspension of any type or any species. Analysis is performed by flow cytometry. Examples of product applications Amplification of weak fluorescence, for example, due to FITC/PE/APC-conjugated antibody staining of antigens which are expressed at low levels, due to immunomagnetic and FITC/PE/APC-conjugated antibody labeling of the same antigen epitope, due to FITC/PE/APC-conjugated antibody staining with a low affinity antibody. Amplification of the fluorescence signal of an FITC/PE/ APC-labeled cell fraction for a clearer flow cytometric discrimination from the non-labeled cell fraction. Amplification of magnetic labeling with Anti-FITC/PE/ APC MicroBeads by increasing the number of Anti-FITC/ PE/APC MicroBead binding sites per cell CD19 APC A B C CD19 APC Faser kit (1x) CD19 Faser Kit (2x) FS FS FS Figure 3: Human peripheral blood mononuclear cells (PBMC) were stained with CD19-APC, human, and the APC fluorescence was increased by two rounds of amplification using the FASER Kit APC. Cells were analyzed by flow cytometry. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence. Product Components Capacity Order no. FASER Kit FITC FASER KIT PE FASER Kit APC 2 2 ml FcR Blocking Reagent 1 ml FITC-Activator (Reagent 1) 1 ml FITC-Enhancer (Reagent 2) 2 2 ml FcR Blocking Reagent 1 ml PE-Activator (Reagent 1) 1 ml PE-Enhancer (Reagent 2) 2 2 ml FcR Blocking Reagent 1 ml APC-Activator (Reagent 1) 1 ml APC-Enhancer (Reagent 2) For 100 For 100 For Viability, conjugation, and staining kits Annexin V Kits and Reagents The Annexin V products are suitable for the identification and enumeration of dead cells, such as apoptotic or necrotic cells, by flow cytometry. Apoptotic cells, which are otherwise undetectable by staining with propidium iodide (PI), can be directly detected through their staining with fluorochrome-conjugated Annexin V. Dead cells are stained with both Annexin V and PI, whereas viable cells cannot be stained with either. In most normal, viable eukaryotic cells, the negatively charged phospholipid phosphatidylserine (PS) is located in the cytosolic leaflet of the plasma membrane lipid bilayer. PS redistribution from the inner to the outer leaflet is an early and widespread event during apoptosis. However, in necrosis, PS becomes accessible due to the disruption of membrane integrity. Apart from necrosis and apoptosis, PS also becomes accessible in activated platelets, in certain cell anomalies like sickle cell anemia, in erythrocyte senescence, upon degranulation of mast cells and in certain stages of B cell differentiation. PS exposure also serves as a trigger for the recognition and removal of apoptotic cells by macrophages. Annexin V is a 35 kda phospholipidbinding protein and a major cell membrane component of macrophages and other phagocytic cell types. Annexin V has a high affinity to PS in the presence of physiological concentrations of calcium (Ca2 + ). Product Capacity/ Content Order no. Annexin V-FITC Kit For Annexin V-FITC For Annexin V-Biotin For Annexin V Binding Buffer 25 ml Annexin V-FITC Extracellular Intracellular Cellular membrane Translocation of PS during apoptosis Staining of PS-exposing cells with Annexin V-FITC Phosphatidylserine (PS) Figure 4: Figure 4:Staining principle of PS-exposing cells with Annexin V-FITC. During necrosis, the negatively charged phospholipid phosphatidylserine (PS) is redistributed from the inner to the outer leaflet of the plasma membrane lipid bilayer, thus becoming accessible due to the disruption of membrane integrity. Necrotic and apoptotic cells, therefore, can be detected following the bining of fluorochromeconjugated Annexin-V.

5 Propidium Iodide Solution The Propidium Iodide Solution is suitable for the exclusion of dead cells from flow cytometric analysis. Propidium iodide (PI) is widely used for staining and evaluation of cell death and apoptosis or for determination of DNA content in cell cycle analysis. PI is a fluorescent dye that intercalates into double-stranded nucleic acid. It is excluded from viable cells, but can penetrate cell membranes of dead or dying cells. Therefore, it is widely used for evaluation of cell death and apoptosis or for determination of DNA content in cell cycle analysis. The fluorescence emission maximum for DNA-bound PI is about nm. When excited by a 488 nm laser, PI can therefore be detected in both, the red fluorescence channel commonly used for R-phycoerythrin (PE)-Cy TM 5 tandem dye detection as well as the yellow fluorescence channel commonly used for R-phycoerythrin (PE) detection. Propidium Iodide Solution 10³ 10² 10¹ P3 1 P ¹ 10² P2 Hoechst Solution Figure 6: Human peripheral blood mononuclear cells (PBMCs) were stained with the Live Cell Discrimination Kit and analyzed by flow cytometry using the MACSQuant Analyzer 10. Cell debris was excluded from the analysis based on scatter signals. P1 shows the debris and erythrocytes, P2 the viable cells, and P3 the PI + dead cells. Product Components Capacity Order no. Live Cell Discrimination Kit 2 ml Hoechst solution 2 ml Propidium Iodide Solution 10³ For Propidium iodide CD90.2-PE P1/P2 Figure 5: 10⁶ mouse splenocytes were stained with MC CD90.2 T Cell Cocktail (# ). Shortly before flow cytometric analysis, 10 μl of Propidium Iodide Solution was added to 1 ml of cell suspension. Cells were analyzed by flow cytometry. Dead cells are positive for PI and thus can be excluded from the analysis. Viable cells belong to the P2 population Product Content Order no. Propidium Iodide Solution 2 ml Live Cell Discrimination Kit The Live Cell Discrimination Kit can be used for the identification and enumeration of live cells using Hoechst to detect nucleated cells and PI staining for the detection of dead cells. Hoechst is a cell permeable nucleic acid dye that intercalates in A-T regions of DNA. Therefore the staining intensity differs between different cell types. All nucleated cells are Hoechst positive. PI is a fluorescent dye that intercalates into double-stranded nucleic acid. It is excluded from viable cells, but can penetrate cell membranes of dead or dying cells. This kit can be used in the exclusion of dead cells and in the enumeration of viable nucleated cells. Fixation and Dead Cell Discrimination Kit The Fixation and Dead Cell Discrimination Kit allows dead cell staining to be combined with cell fixation. The kit contains reagents for fixation and dead cell fluorescent staining and is suitable for cells of any species. Fixation of cells facilitates working with biohazardous samples and also allows the prolonged storage of samples. Before fixation, dead cells are labeled irreversibly with a membrane-impermeable dye that binds covalently to nucleic acid upon exposure to visible light. This staining remains stable after fixation meaning that cells which were dead prior to fixation can be excluded by flow cytometric analysis. Thus, non-specific background staining is significantly reduced and sensitivity of detection enhanced. The Fixation and Dead Cell Discrimination Kit can be used for: Prolonged storage of cell samples (up to 24 hours) Reduced biohazard of infectious samples due to fixation of the cells Highly sensitive analysis of rare cells due to the reduction of background staining caused by dead cells

6 Figure 6: Dead cell discrimination after fixation of fluorescently labeled cells PBMCs were stained with Anti-CRTH2-PE, CD4-FITC, and Dead Cell Discriminator. The sample was exposed to visible light. Cells were washed and fixed, Discriminator Stop Reagent was added to the sample. Lymphocytes were gated according to scatter properties by flow cytometric analysis. Previously dead cells were excluded, in a fluorescence 2 (PE) versus fluorescence 3 (Dead Cell Discriminator) plot, by gating on the fluorescence 3 cells. CRTH2-expression on previously viable, fixed CD4 + lymphocytes is shown. Dead Cell Discriminator Anti-CRTH2-PE Anti-CRTH2-PE Dead Cell Discriminator Dead Cell Discriminator CD4-FITC CD138-PE CD138-PE Figure 7: Dead cell discrimination after fixation of magnetically enriched cells PBMCs were labeled with CD138 MicroBeads, and stained with CD138-PE and Dead Cell Discriminator. After magnetic separation using an MS Column in a MiniMACS Separator, cells were washed, fixed, and Discriminator Stop Reagent was added to the sample. Previously dead cells were excluded by flow-cytometric analysis, in a fluorescence 2 (PE) versus fluorescence 3 (Dead Cell Discriminator) plot, by gating on the fluorescence 3 cells. Product Components Capacity Order no. Fixation and Dead Cell Discrimination Kit 200 μl Dead Cell Discriminator 500 μl Discriminator Stop Reagent 25 ml Fix Solution For Miltenyi Biotec provides products and services worldwide. Visit to find your nearest Miltenyi Biotec contact. MACS, MACSQuant, Vio770, MiniMACS and VioBlue are either registered trademarks or trademarks of Miltenyi Biotec GmbH. Cy is a trademark of GE Healthcare companies. Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. Copyright 2012 Miltenyi Biotec GmbH. All rights reserved.

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