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Identification of 18 Immune Cell Subsets Using 13-Color Panel

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Identification of 18 Immune Cell Subsets Using 13-Color Panel Immunophenotyping Identification of 18 immune cell subsets in human blood using a 13-color panel Background Cell type Function Phenotype Flow cytometry has become the method of choice for Eosinophils Parasitic immunity CD45+, SSCmid/hi, CD14 –, CD16 –, CD19– immunophenotyping and identifying specific cellular + mid/hi subsets. Within seconds, it provides a thorough overview of Neutrophils Innate Immunity CD45 , SSC , CD14 –, CD16+, CD19– the major cell types that constitute a sample. Using multiple + mid markers simultaneously increases the number of parameters Classical Phagocytosis of CD45 , SSC , monocytes pathogens and CD14+, CD16– that can be analyzed per run and decreases the amount of antigen presentation starting material required to perform an assay. This can be Intermediate Phagocytosis of CD45+, SSCmid, critical for precious sample material and long-term immune- monocytes pathogens and CD14+, CD16mid monitoring studies. In this application note, we demonstrate antigen presentation 13-color immunophenotyping of human peripheral blood Non-classical Phagocytosis of CD45+, SSCmid, + + mononuclear cells (PBMCs) using the MACSQuant® Analyzer 16, monocytes pathogens and CD14 , CD16 antigen presentation a compact and reliable benchtop flow cytometer equipped + low + with three lasers. The markers selected allow for the Class-switched Adaptive immunity CD45 , SSC , CD19 , memory B cells CD27+, IgD–, CD14– simultaneous identification and analysis of 18 different cell Non-switched Adaptive immunity CD45+, SSClow, CD19+, populations, thus maximizing the amount of information that memory B cells CD27+, IgD+, CD14– can be retrieved from the sample material analyzed. This is Naive B cells Adaptive immunity – CD45+, SSClow, CD19+, critical when input material is limited, as is often the case for non-antigen CD27–, IgD+, CD14– pediatric or disease studies. experienced Cytotoxic effector Killing of virally CD45+, SSClow, CD19–, memory T cells infected or cancer CD3+, CD4–, CD8+, Materials and methods cells CD45RO+, CD62L–, CD14– PBMCs were labeled with CD14-VioBlue®, CD45-VioGreen™, Cytotoxic central Killing of virally CD45+, SSClow, CD19–, CD8-BV570™, Anti-IgD-BV605™, CD62L-BV650™, memory T cells infected cells CD3+, CD4–, CD8+, CD25-VioBright™ 515, CD27-PE, CD45R0-PE-Vio 615, CD45RO+, CD62L+, – CD4-PerCP-Vio 700, CD19-PE-Vio 770, CD127-APC, CD14 CD3-Alexa Fluor® 700 and CD24-APC-Vio® 770. Data Cytotoxic Killing of virally CD45+, SSClow, CD19–, effector T cells infected cells CD3+, CD4–, CD8+, was acquired on the MACSQuant Analyzer 16 using CD45RO–, CD62L–, MACSQuantify™ Software for acquisition and Flowlogic™ CD14– Software for analysis. The markers used to identify different Cytotoxic naive Killing of virally CD45+, SSClow, CD19–, immune cell populations are described in table 1. T cells infected cells – CD3+, CD4–, CD8+, non-antigen CD45RO–, CD62L+, Cell staining protocol experienced CD14– 1. Resuspend 2×10⁶ leukocytes from lysed whole blood in Effector memory Immune regulation CD45+, SSClow, CD19–, 100 μL of PEB Buffer (phosphate buffered saline, pH 7.2, T helper cells CD3+, CD4+, CD8–, + – 2 mM EDTA, 0.5% BSA). CD45RO , CD62L , CD14– 2. Add conjugated antibodies at vendor recommended Central memory Immune regulation CD45+, SSClow, CD19–, concentrations. T helper cells CD3+, CD4+, CD8–, 3. Incubate for 10 minutes at 4°C. CD45RO+, CD62L+, CD14– 4. Add 1 mL of PEB Buffer to wash cells. 5. Centrifuge at 300×g for 10 minutes and aspirate Table 1: Identification and analysis of PBMC subsets. The table shows a selection of surface markers that can be used for a character- supernatant. ization of immune cell subtypes by flow cytometry. Compiled from 6. Resuspend pellet in 500 μL of PEB Buffer. references 1 and 2. 1 Cell type Function Phenotype Results Effector T helper Immune regulation CD45+, SSClow, CD19–, Figure 1 depicts the gating strategy used to identify target cells CD3+, CD4+, CD8–, CD45RO–, CD62L–, cell populations of interest. By utilizing the expanded CD14– fluorescence capability of the MACSQuant® Analyzer 16, it Naive T helper Immune regulation – CD45+, SSClow, CD19–, is possible to simultaneously characterize the presence of cells non-antigen CD3+, CD4+, CD8–, eosinophils, neutrophils, classical monocytes, intermediate – + experienced CD45RO , CD62L , monocytes, non-classical monocytes, class-switched memory CD14– B cells, non-switched memory B cells, naive B cells, cytotoxic + low – Regulatory T cells Immune suppression CD45 , SSC , CD19 , effector memory T cells, cytotoxic central memory T cells, and regulation CD3+, CD4+, CD8–, CD127–, CD25+, CD14– cytotoxic effector T cells, cytotoxic naive T cells, effector NK cells Viral and cancer cell CD45+, SSClow, CD19–, memory T helper cells, central memory T helper cells, effector clearance CD3–, CD14–, CD16+ T helper cells, naive T helper cells, regulatory T cells, and NK Table 1 (continued): Identification and analysis of PBMC subsets. cells. The data clearly show that the MACSQuant Analyzer 16 The table shows a selection of surface markers that can be used for a enables high-quality flow analysis with a 13-color antibody characterization of immune cell subtypes by flow cytometry. Compiled panel, opening up new possibilities for deeper pheno-typing from references 1 and 2. and immune-monitoring applications. 250 200 150 FSC Singlets 100 97.44% 50 50 100 150 200 250 FSC 250 Leukocytes 250 250 250 98.88% 200 200 200 Monocytes 200 9.62% 150 150 150 150 Morphology B cells SSC SSC SSC SSC 100 100 100 100 5.45% 50 50 50 50 –1 0 1 1e¹ 1e² 1e³ 50 100 150 200 250 –1 0 1 1e¹ 1e² 1e³ –1 0 1 1e¹ 1e² 1e³ CD45-VioGreen FSC CD14-VioBlue CD19-PE-Vio 770 1e³ 1e³ Non-switched memory 250 Eosionophils B cells 16.39% 1.12% 1e² 1e² Class switched memory 200 Non-classical B cells 32.28% monocytes 11.53% 150 Neutrophils 80.66% 1e¹ 1e¹ SSC Inter- 100 mediate CD27-PE mono- 1 Classical 1 50 CD16-APC-Vio 770 cytes 0 monocytes 0 Naive B cells 7.41% 46,08% –1 76.31% –1 –1 0 1 1e¹ 1e² 1e³ –1 0 1 1e¹ 1e² 1e³ –1 0 1 1e¹ 1e² 1e³ CD14-VioBlue IgD-BV605 CD16-APC-Vio 770 1e³ 1e² T cells 76.67% 1e¹ 1 NK cells CD3-Alexa Fluor 700 Fluor CD3-Alexa 0 11.73% –1 –1 0 1 1e¹ 1e² 1e³ CD16-APC-Vio 770 1e³ 1e³ 1e³ 1e³ Naive Cytotoxic T cells naive T cells H Central memory Central memory Treg 14.91% 25.28% 1e² 1e² T cells 37.32% 1e² T cells 1.60% 1e² + cells H H CD8 cells 8.12% 24.84% 1e¹ 1e¹ 1e¹ 1e¹ CD8-BV570 1 CD62L-BV650 1 Effector CD62L-BV650 1 Cytotoxic Cytotoxic effector 1 Effector memory CD4+ T cells CD25-VioBright 515 CD25-VioBright effector memory T cells 0 0 TH cells 0 0 TH cells 45.59% 70.29% –1 –1 2.17% –1 T cells 14.61% 48.52% –1 –1 0 1 1e¹ 1e² 1e³ –1 0 1 1e¹ 1e² 1e³ –1 0 1 1e¹ 1e² 1e³ –1 0 1 1e¹ 1e² 1e³ CD127-APC CD45RO-PE-Vio 615 CD45RO-PE-Vio 615 CD4-PerCP-Vio 700 Figure 1: Gating strategy for identification of the target cell populations. 2 Conclusions Product Clone Order no. This application note demonstrates the utility of flow Miltenyi Biotec products cytometry for the detection and enumeration of 18 cellular CD14-VioBlue® REA599 130-110-524 subsets found in human blood using a 13-color antibody CD45-VioGreen™ REA747 130-110-638 panel. CD27-PE M-T271 130-113-630 • Save 50% of sample material by reducing the need to CD45RO-PE-Vio® 615 REA611 130-113-562 split panels into a second tube/well* CD4-PerCP-Vio 700 REA623 130-113-228 • Use up to 40% less reagents* CD19-PE-Vio 770 REA675 130-113-647 • In addition to population statistics, you also gain cell CD127-APC REA614 130-113-413 concentration figures for each of the 18 cell subsets analyzed CD16-APC-Vio 770 REA423 130-113-390 *compared to an 8-color flow cytometry assay CD25-VioBright® 515 REA570 Coming soon Red Blood Cell Lysis Solution 130-094-183 References Others CD3-Alexa Fluor® 700 OCT3 1. Maecker, T.H. et al. (2012) Nature Immunol. 12: 191–200. 2. Olingy, C.E. et al. (2017) Sci. Rep. 7: 447. CD8-Brilliant Violet 570™ RPA-T8 IgD-Brilliant Violet 605™ IA6-2 CD62L-Brilliant Violet 650™ Dreg-56 Miltenyi Biotec GmbH | Phone +49 2204 8306-0 | Fax +49 2204 85197 | [email protected] | www.miltenyibiotec.com Miltenyi Biotec provides products and services worldwide. Visit www.miltenyibiotec.com/local to find your nearest Miltenyi Biotec contact. Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. MACS, the MACS logo, MACSQuant, Vio, VioBlue, VioBright, and VioGreen are registered trademarks or trademarks of Miltenyi Biotec GmbH and/or its affiliates in various countries worldwide. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Copyright © 2018 Miltenyi Biotec GmbH and/or its affiliates. All rights reserved. V. 02.
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