ab pdc Subset Phenotyping Kit

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1 ab pdc Subset Phenotyping Kit Instructions for Use A set of antibodies designed to identify pdcs and subsets of pdcs by multi-color flow cytometry. This product is for research use only and is not intended for diagnostic use. Version 4 Last Updated 25 July 2014

2 Table of Contents 1. Product Overview 2 2. Protocol Summary 4 3. Materials Supplied 5 4. Storage 6 5. Materials Required, Not Supplied 6 6. General Considerations 7 7. Protocol 8 8. Typical Data 10 1

3 1. Product Overview pdcs are identified by Flow Cytometry using lineage markers HLADR and CD123 antigens as unique markers. Recent investigations identified and established two additional markers for pdcs, such as ILT7 (CD85g) and BDCA2. Abcam s pdc Subset Phenotyping Kit consists of a set of antibodies as selected markers conjugated to different fluorochromes. It is designed to identify pdcs and subsets of pdcs by multi-color flow cytometry. The kit offers a facilitated and more precise identification of pdcs, directly from whole blood or PBMC with minimal manipulation and a tool for monitoring pdc subset changes during chronic viral infections or diseases. The pdc Subset Phenotype Kit is validated to identify subsets of pdcs from whole blood and PBMC. pdcs from peripheral blood play an important role in both innate and adaptive immune responses although present in small numbers. 2

4 Some pdc functions include: Recognition of viral components, such as nucleic acids, via Toll receptors TLR7 and TLR9 Production of Type 1 IFNα as a primary anti-viral immune response Antigen presentation as APCS- a critical functional component eliciting adaptive immunity 3

5 2. Protocol Summary Note: All staining and incubation steps should be done using light protection and dark. Prepare blood or PBMC suspension Add the antibodies and mix well Prepare isotype control (optional) For PBMCs: Incubate on ice in the dark For RBCs: Incubate at room temperature and lyse before incubating again Add cold staining buffer. Gently mix Centrifuge Discard supernatant and repeat wash Suspend and analyze by flow cytometry 4

6 3. Materials Supplied Item Isotype Quantity Mouse monoclonal [L243] to HLA DR (PerCP/Cy5.5 ) IgG2a 5 µl Mouse monoclonal [6H6] to CD123 (PE/Cy7 ) IgG1 5 µl Mouse monoclonal [101C9] to CD303/BDCA-2 (PE) IgG1 5 µl Mouse monoclonal [17G10.2] to ILT7/CD85g (AlexaFluor 647) Lineage Marker Antibody Mix (LMAX) Contains: IgG1 5 µl 10 µl Mouse monoclonal to CD3 (FITC) IgG2a Mouse monoclonal to CD14 (FITC) IgG1 Mouse monoclonal to CD16 (FITC) IgG1 Mouse monoclonal to CD19 (FITC) IgG1 Mouse monoclonal to CD20 (FITC) IgG2b Mouse monoclonal to CD56 (FITC) IgG1 5

7 4. Storage Store all kit components at 4 C. 5. Materials Required, Not Supplied Staining buffer Red blood cell lysis buffer Disposable tubes Vortex 6

8 6. General Considerations 1. All staining and incubation steps should be done using light protection and dark. 2. Unstained and single color control tubes are useful for instrument set-up and compensation 3. Reagents for compensation and isotype controls are not provided in this kit (Details (including isotype) of each kit component are mentioned in section 3). 4. Researcher may use FMO (Fluorescence Minus One) controls to identify low numbers of positive population. 5. Read the application and testing protocol before starting the experiment. For further technical questions please do not hesitate to contact us by or phone (select contact us on for the phone number for your region). 7

9 7. Protocol Staining of PBMC and whole blood to identify subset(s) of pdc is a straight forward and a standard protocol, as briefly described below. 1. Determine the number of cells required for staining which include cells for staining as well as cells for unstained control. Note: 1 x 10 6 cells per each staining sample generally needed. 2. Add 100 µl of blood or PBMC suspension (1 x 10 6 cells in 100 µl) into a labeled tube. 3. Add the antibodies according to the following table and mix well. Antibody Quantity Mouse monoclonal [L243] to HLA DR (PerCP/Cy5.5 ) 5 µl Mouse monoclonal [6H6] to CD123 (PE/Cy7 ) 5 µl Mouse monoclonal [101C9] to CD303/BDCA-2 (PE) 5 µl Mouse monoclonal [17G10.2] to ILT7/CD85g (AlexaFluor 647) 5 µl Lineage Marker Antibody Mix (LMAX) 10 µl 8

10 Note: Single color (AlexaFluor 647, FITC, PE, PE/Cy7 and PerCP/Cy5.5 ) stained samples are recommended as compensation controls for flow cytometric analysis. 4. For PBMC, incubate for 30 min on ice. For whole blood, incubate for 20 min at room temperature (RT) in dark. After incubation, add 2 ml of red blood cell lysis buffer and continue incubation for 10 more minutes at RT. Note: All subsequent steps are common to both PBMC and whole blood samples. 5. Add 2 ml of cold staining buffer. Gently vortex the tubes to mix the contents 6. Centrifuge for 10 min at 1200 RPM. 7. Aspirate/decant the supernatant being careful not to lose the cells. 8. Repeat Steps 5-7 to wash. 9

11 9. Suspend the pellet in 300 µl of staining buffer and analyze by flow cytometry. Note: When not analyzing on the same day, suspend cells in fixation buffer and store overnight at 4 C. The fixation buffer can be removed and the cells prepared for analysis by repeating Step 7-9 and adding 300 µl of staining buffer to each tube. 8. Typical Data Figure 1. pdc identified as Lin-DR + CD123 + cells were further analyzed based on expression of ILT7 and BDCA2. Panel A: Gate Lineage negative HLA-DR population Panel B: Identify pdc as HLA-DR and CD123 double positive cells and gate them Panel C: Analyze subsets of pdc using markers ILT7 (CD85g) and BDCA2 (CD303) as shown 10

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