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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

This work was supported by the National Natural Science Foundation of China (82002130), Beijing Natural Science Foundation (7222059) and the CAMS Innovation Fund for Medical Sciences (2019-I2M-5-026).

....

Materials

NameCompanyCatalog NumberComments
APC anti-human CD16Biolegend302012Fluorescent antibody 
APC anti-human CD56 (NCAM)Biolegend362504Fluorescent antibody 
APC/Cyanine7 anti-human CD4Biolegend344616Fluorescent antibody 
Automated cell counterBIO RADTC20Cell count
BD FACSAria fluorescence-activated flow cell sorter-cytometer (BD FACSAria II)Becton, Dickinson and Company644832Cell sort
CD14 MicroBeads, humanMiltenyi Biotec130-050-201microbeads
Cell ctng slidesBIO RAD1450016Cell count
Centrifuge tubesFalcon3520971515 mL centrifuge tube
EDTA (≥99%, BioPremium)BeyotimeST1303EDTA
Ethylene diamine tetra acetic acid (EDTA) anticoagulant tubesBecton, Dickinson and Company 367862 EDTA anticoagulant tubes
FITC anti-human CD19Biolegend302206Fluorescent antibody 
Gibco Fetal Bovine SerumThermo Fisher Scientific16000-044Fetal Bovine Serum
 High-speed centrifugeSigma 3K15Cell centrifugation for 15 mL centrifuge tube
 High-speed centrifugeEppendorf5424RCell centrifugation for 1.5 mL Eppendorf (EP) tube
Human lymphocyte separation mediumDakeweDKW-KLSH-0100Ficoll-Paque
LS Separation columnsMiltenyi Biotec130-042-401Separation columns
Microcentrifuge tubesAxygenMCT-150-C1.5 mL microcentrifuge tube
MidiMACS SeparatorMiltenyi Biotec130-042-302Magnetic bead separator
PE anti-human CD8Biolegend344706Fluorescent antibody 
PerCP/Cyanine5.5 anti-human CD3Biolegend344808Fluorescent antibody 
Phosphate Buffered Saline (PBS)BI02-024-1ACSPBS
Polystyrene round bottom tubesFalcon3522355 mL tube for FACS flow cytometer
TRIzol reagentAmbion15596024Lyse cells for RNA extraction
Trypan Blue Staining Cell Viability Assay KitBeyotimeC0011Trypan Blue Staining

References

  1. Kwok, H., Chiang, A. K. From conventional to next generation sequencing of Epstein-Barr virus genomes. Viruses. 8 (3), 60 (2016).
  2. Bu, W., et al.

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Immune Cell SubpopulationsPeripheral Blood SamplesInfectious MononucleosisPBMC IsolationCD14 MicrobeadsMagnetic SeparationTranscriptome SequencingFluorescent Antibody StainingFlow Cytometry

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