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We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.
Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein–Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.
Epstein–Barr virus (EBV), a γ-herpesvirus also known as human herpes virus type 4, is ubiquitous in the human population and establishes lifelong latent infection in more than 90% of the adult population1. Most EBV primary infection occurs during childhood and adolescence, with a fraction of patients manifesting with infectious mononucleosis (IM)2, showing characteristic immunopathology, including an activated immune response with CD8+ T cells in blood and a transient proliferation of EBV-infected B cells in the oropharynx3. The course of IM may last for 2–6 weeks and t....
Blood samples were obtained from patients with IM (n = 3), healthy EBV carriers (n = 3), and EBV-uninfected children (n = 3). Volunteers were recruited from Beijing Children's Hospital, Capital Medical University, and all studies were ethically approved. Ethical approval was obtained by the Ethics Committee of Beijing Children's Hospital, Capital Medical University (Approval Number: [2021]-E-056-Y). Informed consent of patients was waived as the study only used the remaining samples for clinical testing. All data.......
Reference of the gating strategy
The gating strategy used to sort the four lymphocyte subpopulations is shown in Figure 1. Briefly, lymphocytes are selected (P1) on a dot plot showing the granulosity (SSC-A) versus size (FSC-A). Then, single cells are selected (P2) on a dot plot showing the size (FSC-A) versus forward scatter (FSC-H), while doublet cells are excluded. CD3+ T cells (P3) and CD19+ B cells (Figure 1B) ar.......
This protocol represents an efficient way to sort peripheral blood immune cell subpopulations. In this study, venous blood samples from patients with IM, healthy EBV carriers, and EBV-uninfected children were selected as the research objective. This work using the peripheral blood of patients of IM mainly focuses on analyzing and determining the proportions of different cell subsets through multi-color flow cytometry. Transcriptome sequencing is mainly used for the detection and analysis of a certain subpopulation of lym.......
This work was supported by the National Natural Science Foundation of China (82002130), Beijing Natural Science Foundation (7222059)Â and the CAMS Innovation Fund for Medical Sciences (2019-I2M-5-026).
....Name | Company | Catalog Number | Comments |
APC anti-human CD16 | Biolegend | 302012 | Fluorescent antibody |
APC anti-human CD56 (NCAM) | Biolegend | 362504 | Fluorescent antibody |
APC/Cyanine7 anti-human CD4 | Biolegend | 344616 | Fluorescent antibody |
Automated cell counter | BIO RAD | TC20 | Cell count |
BD FACSAria fluorescence-activated flow cell sorter-cytometer (BD FACSAria II) | Becton, Dickinson and Company | 644832 | Cell sort |
CD14 MicroBeads, human | Miltenyi Biotec | 130-050-201 | microbeads |
Cell ctng slides | BIO RAD | 1450016 | Cell count |
Centrifuge tubes | Falcon | 35209715 | 15 mL centrifuge tube |
EDTA (≥99%, BioPremium) | Beyotime | ST1303 | EDTA |
Ethylene diamine tetra acetic acid (EDTA) anticoagulant tubes | Becton, Dickinson and Company | Â 367862Â | EDTA anticoagulant tubes |
FITC anti-human CD19 | Biolegend | 302206 | Fluorescent antibody |
Gibco Fetal Bovine Serum | Thermo Fisher Scientific | 16000-044 | Fetal Bovine Serum |
 High-speed centrifuge | Sigma |  3K15 | Cell centrifugation for 15 mL centrifuge tube |
 High-speed centrifuge | Eppendorf | 5424R | Cell centrifugation for 1.5 mL Eppendorf (EP) tube |
Human lymphocyte separation medium | Dakewe | DKW-KLSH-0100 | Ficoll-Paque |
LS Separation columns | Miltenyi Biotec | 130-042-401 | Separation columns |
Microcentrifuge tubes | Axygen | MCT-150-C | 1.5 mL microcentrifuge tube |
MidiMACS Separator | Miltenyi Biotec | 130-042-302 | Magnetic bead separator |
PE anti-human CD8 | Biolegend | 344706 | Fluorescent antibody |
PerCP/Cyanine5.5 anti-human CD3 | Biolegend | 344808 | Fluorescent antibody |
Phosphate Buffered Saline (PBS) | BI | 02-024-1ACS | PBS |
Polystyrene round bottom tubes | Falcon | 352235 | 5 mL tube for FACS flow cytometer |
TRIzol reagent | Ambion | 15596024 | Lyse cells for RNA extraction |
Trypan Blue Staining Cell Viability Assay Kit | Beyotime | C0011 | Trypan Blue Staining |
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