Name: separation of immune cell subpopulations in peripheral blood samples from children with infectious mononucleosis
Uploaded: 2022-09-07T13:00:00.000+00:00
Duration: 8 min 44 s
Description: Watch this Scientific Journal Video about Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis at JoVE.com

This work was supported by the National Natural Science Foundation of China (82002130), Beijing Natural Science Foundation (7222059)&#160;and the CAMS Innovation Fund for Medical Sciences (2019-I2M-5-026).

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&#39;s Hospital, Capital Medical University, and all studies were ethically approved. Ethical approval was obtained by the Ethics Committee of Beijing Children&#39;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 were fully deidentified and anonymized to protect patient privacy.
1. Isolation of PBMCs from peripheral blood
<ol>
	<li>Collect fresh peripheral blood (2 mL) from patients with IM into K3EDTA tubes by standard venipuncture. 
	NOTE: The process needs to be rapid to maintain cell viability.</li>
	<li>Dilute peripheral blood to twofold volume with phosphate-buffered saline (PBS) and layer it on top of human lymphocyte separation medium (density: 1.077 &#177; 0.001 g/mL) in a 15 mL centrifuge tube. 
	NOTE: The volume ratio of blood, PBS, and separation medium was 1:1:1. Add the blood slowly to the separation medium, and centrifuge immediately to avoid blood settling into the separation medium.</li>
	<li>Centrifuge at 800 &#215; g for 20 min at room temperature. Transfer the middle layer (enriched PBMCs) to another 15 mL centrifuge tube. 
	NOTE: After centrifugation, at the bottom of the tube are erythrocytes, the middle layer is the separation medium, the top layer is plasma, and between the plasma layer and the separation liquid layer were the PBMCs (including lymphocytes and monocytes).</li>
	<li>Wash the PBMCs with 10 mL of PBS and centrifuge at 800 &#215; g for 20 min at room temperature; discard the supernatant carefully.</li>
	<li>Repeat the washing and centrifuging (step 1.4) 2 x. Resuspend the PBMCs with 1 mL of PBS in a 1.5 mL microcentrifuge tube and count the cells with a trypan blue-based, automated counter.</li>
</ol>
2. Isolation of CD14&#160;+ monocytes from PBMCs using CD14 microbeads
<ol>
	<li>Prepare a buffer solution containing 0.5% fetal bovine serum (FBS) and 2 mM EDTA in PBS (pH 7.2). Keep the buffer cold (2&#8722;8 &#176;C). 
	NOTE: Degas the buffer before use as air bubbles could block the column. Keep the cells cold to prevent capping of the antibodies on the cell surface and non-specific cell labeling.</li>
	<li>Centrifuge the PBMCs at 300 &#215; g for 10 min at room temperature. Discard the supernatant carefully. Resuspend the cells with 80 &#181;L of the buffer. Add 20 &#181;L of CD14 microbeads to the cell suspension. 
	NOTE: If there are &#8804;107 PBMCs, use the volume indicated above. If there are &#62;107 PBMCs, proportionally increase all reagent volumes and the total volume.</li>
	<li>Mix the CD14 microbeads and the cells well in the 1.5 mL microcentrifuge tube and incubate for 15 min in a 4 &#176;C refrigerator. Wash the PBMCs with 1 mL of buffer and centrifuge at 300 &#215; g for 10 min at room temperature. Discard the supernatant completely. Resuspend the cells with 500 &#181;L of the buffer. 
	NOTE: If there are &#8804;108 PBMCs, use the volume indicated above. If there are &#62;108 PBMCs, proportionally increase the buffer volume.</li>
	<li>Magnetic separation with columns:
	<ol>
		<li>Put the column on the magnetic bead separator, and wash the column with 3 mL of buffer. Add the cell suspension (from step 2.3) to the column.</li>
		<li>Collect the unlabeled cells that pass through the column into a 15 mL centrifuge tube and wash the column with 3 mL of buffer. Wash the column with 3 x 3 mL of buffer. Collect the total effluent in the 15 mL centrifuge tube.</li>
		<li>Place the column in a new 15 mL centrifuge tube. Add 5 mL of buffer to the column. Push the plunger firmly into the column to immediately flush out the magnetically labeled cells into the 15 mL centrifuge tube.</li>
		<li>Centrifuge the magnetically labeled cells at 300 &#215; g for 5 min and remove the supernatant. Resuspend the cells in 500 &#181;L of PBS in a 1.5 mL microcentrifuge tube for use in subsequent transcriptome sequencing.</li>
	</ol>
	</li>
</ol>
3. Separation of lymphocyte populations from PBMCs by fluorescently labeled antibody staining and FACS
<ol>
	<li>Centrifuge the unlabeled cells (step 2.4.2) at 300 &#215; g for 5 min and remove the supernatant. Resuspend the cells in 100 &#181;L of PBS in a 1.5 mL microcentrifuge tube.</li>
	<li>Add 2 &#181;L of each labeled antibody (CD3, CD4, CD8, CD16, CD19, CD56) to the 100 &#181;L of cell suspension (the volumes and conjugated fluorophore information of antibodies are shown in Table 1), incubate on ice for 30 min, and protect from light.</li>
	<li>Wash the cells 2 x by adding 1 mL of PBS and centrifuging at 300 &#215; g for 5 min at room temperature. Resuspend the cells in 500 &#181;L of PBS in the 1.5 mL microcentrifuge tube. Vortex the cell suspension gently before acquiring data on a flow cell sorter cytometer.</li>
</ol>
4. Flow cytometry parameter setting
<ol>
	<li>Take 100 &#181;L of the cell suspension (see section 1) in 1.5 mL microcentrifuge tubes as required, and set up a negative control sample, a CD3 single-staining sample, a CD4 single-staining sample, a CD8 single-staining sample, a CD19 single-staining sample, and a CD56/CD16 staining sample.</li>
	<li>Add 2 &#181;L of the corresponding fluorescently labeled antibody per 100 &#181;L of the cell suspension and vortex. Incubate on ice for 30 min in the dark. Centrifuge for 5 min at 300 &#215; g and aspirate the supernatant. Resuspend the pellets with 500 &#181;L of PBS and vortex.</li>
	<li>Commission the sorting stream and delay the droplets using the fluorescent beads as follows:
	<ol>
		<li>Open the cell sorting system, run the power-on program, install the 85 &#181;m nozzle, and open the sorting stream. Set the sorting voltage to 4,500 V, and the Freq to 47.</li>
		<li>Adjust the parameters mainly by adjusting the main flow droplet breakpoint and the droplet delay according to the manufacturer&#39;s instructions12. Set up the first droplet breakpoint position (Drop1) to 275, and the Gap to 8 in the Breakoff window. Turn on the Sweet Spot automatic sorting mode to allow the cytometry to automatically determine the droplet amplitude value to stabilize the stream.</li>
		<li>Adjust the droplet delay to 30.31 in the Side Stream window by loading the fluorescent beads, ensuring that the beads achieve a side flow deflection of &#62;99% in either initial or fine tune mode.</li>
	</ol>
	</li>
	<li>Refer to the gating strategy shown in Figure 1 to perform gating as follows:
	<ol>
		<li>Using a forward scatter-area (FSC-A)/side scatter-area (SSC-A) dot plot, draw the polygon gate (P1) to identify the intact lymphocyte population.</li>
		<li>Using an FSC-A/FSC-height (FSC-H) dot plot, draw the polygon gate (P2) to identify the single cells and exclude doublets (Figure 1A).</li>
		<li>Using a CD19 FITC-A/CD3 PerCP-Cy5.5-A dot plot, draw the rectangular gate (P3) to select CD3+ T cells and CD19+ B cells (Figure 1A,B).</li>
		<li>Using a CD4 APC-Cy7-A/CD8 PE-A dot plot, draw a rectangular gate to select CD4+ T cells and CD8+ T cells (cells with a high fluorescence for these markers, respectively).</li>
		<li>Using a CD56/CD16 APC-A/SSC-A dot plot, draw a rectangular gate to select CD56+/CD16+ NK cells (Figure 1C).</li>
	</ol>
	</li>
	<li>Adjust instrumental parameters using the negative control sample:
	<ol>
		<li>Install the negative control tube onto the loading port and click Load in the Acquisition Dashboard. Select the Cytometer Settings in the software.</li>
		<li>In the Inspector window, click the Parameters tab, and adjust the voltages of FSC, SSC, and different fluorescent dyes; FSC: 231, SSC: 512, PE: 549, APC: 615, APC-Cyanine 7: 824, FITC: 555, PerCP-Cyanine 5.5: 663.</li>
	</ol>
	</li>
	<li>Adjust compensation using the single-stained samples13.
	<ol>
		<li>Load the single-stained tubes onto the cytometry sequentially andselect Cytometer Settings in the software. Click the Compensation tab to adjust the compensation. 
		&#8203;NOTE: The compensation reference of flow cytometry is shown in Table 2.</li>
	</ol>
	</li>
</ol>
5. Cell sorting and collecting data&#160;&#160;via flow cytometry
<ol>
	<li>Vortex the cell suspension (separated PBMCs according to sections 1&#8211;3) briefly to resuspend the cells before loading the tube into the cytometer. Keep the remaining tubes on ice.</li>
	<li>Add 200 &#181;L of FBS to four collection flow tubes to avoid sticking of the sorted cells to the tube wall and place them in the cytometer collection chamber.</li>
	<li>Collect CD3+CD4+ T cells, CD3+CD8+ T cells, CD3&#8722;CD19+ B cells, and CD3&#8722; CD56+/CD16+ NK cells separately from the sample of a patient with IM in the four flow tubes (Figure 2A).</li>
	<li>Centrifuge the separated cells at 300 &#215; g for 5 min and remove the supernatant. Add 200 &#181;L of RNA isolation reagent to the cells for transcriptome sequencing.</li>
	<li>Separate the immune cell subpopulations of samples from healthy EBV carriers and EBV-uninfected children according to the above steps (Figure 2B, C).</li>
</ol>

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The authors declare no conflicts of interest.

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 lymphocytes that was insufficient for the comprehensive and specific comparisons of the molecular characteristics and functions of different immune cell subpopulations in children with IM at the same disease state. Therefore, sorting out several types of cells from the peripheral blood and performing transcriptome sequencing to compare the differences in the expression genes and functions of these immune cells could provide significant data for the study of the pathogenesis of IM.
FACS sorting has the additional benefit of being able to process live, fractionated cells for further in vitro or in vivo experiments14. Maintaining the cell viability is vital for subsequent experiments. We have optimized the sorting step to improve cell viability in this protocol-experimental manipulations have been performed on ice or in a 4 &#176;C refrigerator. Proper centrifugation speed and time are also critical for cell isolation. The yield of sorted cells is also the main constraint in this method, and the use of FBS-coated tubes during sorting can greatly reduce the loss of cells that adhere to the tubes. Providing the proper settings for the FACS sorter can improve the overall efficiency of the sorting by avoiding cell blockage or cross-contamination in the collection tube. Through the optimization of experimental steps and settings, this protocol can be extrapolated to the sorting of other immune cells by replacing magnetic or fluorescent labels. However, due to the specificity of the children&#39;s samples (low blood collection volume), we only investigated the major populations of immune cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and NK cells) without proceeding to sort subpopulations due to the restriction of the flow sorting channel. This study demonstrated that peripheral blood samples from immunocompetent children and samples from IM could successfully sort out these five groups of immune cells; however, the blood samples of patients with hematological malignancies (e.g., leukemia, lymphoma), lymphoproliferative disorders (e.g., posttransplant lymphoproliferative disorders, CAEBV), or primary immunodeficiency/acquired immune deficiency syndrome may not be successfully sorted according to this protocol.
IM is considered a self-limiting disease, and immune cells such as &#947;&#948; T cells, NKT cells, and NK cells play a significant role in the antiviral immune response15. EBV-specific CD8+ T cells are largely differentiated toward an effector phenotype10,16, and there is contraction of late effector memory and effector cells from IM to convalescence17. Meanwhile, NK cells in IM appear to be functionally defective, including lack of cell activation10, loss of activating receptor signaling, and degranulation18. Some studies have found that CD4+ T cells can not only assist CD8+ T cells to kill and eliminate EBV-infected B cells but also inhibit the proliferation of B cells by secreting cytokines and even directly play a killing role during EBV infection19,20. As shown in this study, an increased proportion of CD3+ CD8+ T cells was observed in patients with IM compared to both healthy EBV carriers and EBV-uninfected children. In contrast, decreased proportions of CD3+ CD4+ T cells, CD19+ B cells, and CD16+/CD56+ NK cells were observed in patients with IM compared with EBV-uninfected children. However, the function and gene expression of these different subtypes of immune cells in the same disease state of IM remain unclear. Further analysis of the gene expression profiles of specific immune cell subsets in IM can help to gain insight into the pathogenic mechanism of IM.
We provide a strategy that combines immunomagnetic bead sorting and FACS to isolate and analyze defined immune cell subpopulations in PBMCs. Monocytes are separated first using CD14 microbeads, and the remaining PBMCs are stained with corresponding fluorescently labeled antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells through FACS. We further used these sorted cells for RNA extraction and transcriptome sequencing to characterize the function and gene expression of different immune cells in the immunopathology of IM. The purity and yield of the sorted cells was generally sufficient to conduct gene expression studies (data not shown). Therefore, the sorting method of separate immune cell subpopulations can be used to further explore EBV-associated lymphoproliferative disorders such as CAEBV, post-transplant lymphoproliferative disorder to detect pathogenic genes, pathogenic proteins, and potential therapeutic targets.

Epstein&#8211;Barr virus (EBV), a &#947;-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&#8211;6 weeks and the majority of the patients recover well4. However, some individuals develop persistent or recurrent IM-like symptoms with high morbidity and mortality, which is classified as chronic active EBV infection (CAEBV)5. In addition, EBV is an important oncogenic virus, which is closely related to a variety of malignancies, including epithelioid and lymphoid malignancies such asnasopharyngeal carcinoma, Burkitt&#39;s lymphoma, Hodgkin&#39;s lymphoma (HL), and T/NK cell lymphoma6. Although EBV has been studied for over 50 years, its pathogenesis and the mechanism by which it induces the proliferation of lymphocytes have not been fully elucidated.
Several studies have investigated the molecular signatures for the immunopathology of EBV infection by transcriptome sequencing. Zhong et al. analyzed whole-transcriptome profiling of peripheral blood mononuclear cells (PBMCs) from Chinese children with IM or CAEBV to find that CD8+ T cell expansion was predominantly found in the IM group7, suggesting that CD8+ T cells may play a major role in IM. Similarly, another study found lower proportions of EBV-specific cytotoxic T and CD19+ B cells and higher percentages of CD8+&#160;T cells in patients with IM caused by primary EBV infection than in patients with IM caused both by EBV reactivation and other agents8. B cells are first involved in IM because EBV receptors are presented on their surface. Al Tabaa et al. found that B cells were polyclonally activated and differentiated intoplasmablasts (CD19+, CD27+ and CD20&#8722;, and CD138&#8722; cells) and plasma cells (CD19+, CD27+ and CD20&#8722;, and CD138+) during IM9. Moreover, Zhong et al. found that&#160;monocyte markers CD14 and CD64 were upregulated in CAEBV, suggesting that monocytes may play an important role in the cellular immune response of CAEBV through antibody-dependent cellular cytotoxicity (ADCC) and hyperactive phagocytosis7. Alka&#160;et al. characterized the transcriptome of MACS sorted CD56dim CD16+ NK cells from four patients of IM or HL and found that NK cells from both IM and HL had downregulated innate immunity and chemokine signaling genes, which could be responsible for the hyporesponsiveness of NK cells10. In addition, Greenough et al. analyzed gene expression of sorted CD8+ T cells from 10 PBMCs of individuals with IM. They reported that a large proportion of CD8+ T cells in IM were virus-specific, activated, dividing, and primed to exert effector activities11. Both T cell-mediated, EBV-specific responses, and NK cell-mediated, nonspecific responses play essential roles during primary EBV infection. However, these studies only investigated the transcriptome results of the diverse mixture of immune cells or only a certain subpopulation of lymphocytes, which is not sufficient for the comprehensive comparison of the molecular characteristics and functions of different immune cell subpopulations in children with IM at the same disease state.
This paper describes a method that combines immunomagnetic beads and fluorescence-activated cell sorting (FACS) to isolate and analyze defined immune cell subpopulations of PBMCs (monocytes, CD4+ T cells, CD8+ T cells, B cells, and NK cells). Using this method, magnetic and fluorescently labeled cells can be purified using a magnetic separator and FACS or analyzed by flow cytometry. RNA can be extracted from the purified cells for transcriptome sequencing. This method will enable the characterization and gene expression of different immune cells in the same states of disease of individuals with IM, which will expand our understanding of the immunopathology of EBV infection.

<table><tbody><tr><td>APC anti-human CD16</td><td>Biolegend</td><td>302012</td><td>Fluorescent antibody&amp;nbsp;</td></tr><tr><td>APC anti-human CD56 (NCAM)</td><td>Biolegend</td><td>362504</td><td>Fluorescent antibody&amp;nbsp;</td></tr><tr><td>APC/Cyanine7 anti-human CD4</td><td>Biolegend</td><td>344616</td><td>Fluorescent antibody&amp;nbsp;</td></tr><tr><td>Automated cell counter</td><td>BIO RAD</td><td>TC20</td><td>Cell count</td></tr><tr><td>BD FACSAria fluorescence-activated&amp;nbsp;flow&amp;nbsp;cell&amp;nbsp;sorter-cytometer (BD FACSAria II)</td><td>Becton, Dickinson and Company</td><td>644832</td><td>Cell sort</td></tr><tr><td>CD14 MicroBeads, human</td><td>Miltenyi Biotec</td><td>130-050-201</td><td>microbeads</td></tr><tr><td>Cell ctng slides</td><td>BIO RAD</td><td>1450016</td><td>Cell count</td></tr><tr><td>Centrifuge tubes</td><td>Falcon</td><td>35209715</td><td>15 mL centrifuge tube</td></tr><tr><td>EDTA (&amp;ge;99%, BioPremium)</td><td>Beyotime</td><td>ST1303</td><td>EDTA</td></tr><tr><td>Ethylene diamine tetra acetic acid (EDTA) anticoagulant tubes</td><td>Becton, Dickinson and Company</td><td>&amp;nbsp;367862&amp;nbsp;</td><td>EDTA anticoagulant tubes</td></tr><tr><td>FITC anti-human CD19</td><td>Biolegend</td><td>302206</td><td>Fluorescent antibody&amp;nbsp;</td></tr><tr><td>Gibco Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>16000-044</td><td>Fetal Bovine Serum</td></tr><tr><td>&amp;nbsp;High-speed&amp;nbsp;centrifuge</td><td>Sigma</td><td>&amp;nbsp;3K15</td><td>Cell centrifugation for 15 mL centrifuge tube</td></tr><tr><td>&amp;nbsp;High-speed&amp;nbsp;centrifuge</td><td>Eppendorf</td><td>5424R</td><td>Cell centrifugation for 1.5 mL Eppendorf (EP) tube</td></tr><tr><td>Human lymphocyte separation medium</td><td>Dakewe</td><td>DKW-KLSH-0100</td><td>Ficoll-Paque</td></tr><tr><td>LS&amp;nbsp;Separation&amp;nbsp;columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td>Separation&amp;nbsp;columns</td></tr><tr><td>Microcentrifuge tubes</td><td>Axygen</td><td>MCT-150-C</td><td>1.5 mL microcentrifuge tube</td></tr><tr><td>MidiMACS Separator</td><td>Miltenyi Biotec</td><td>130-042-302</td><td>Magnetic bead separator</td></tr><tr><td>PE anti-human CD8</td><td>Biolegend</td><td>344706</td><td>Fluorescent antibody&amp;nbsp;</td></tr><tr><td>PerCP/Cyanine5.5 anti-human CD3</td><td>Biolegend</td><td>344808</td><td>Fluorescent antibody&amp;nbsp;</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>BI</td><td>02-024-1ACS</td><td>PBS</td></tr><tr><td>Polystyrene round bottom tubes</td><td>Falcon</td><td>352235</td><td>5 mL tube for FACS flow cytometer</td></tr><tr><td>TRIzol reagent</td><td>Ambion</td><td>15596024</td><td>Lyse cells for RNA extraction</td></tr><tr><td>Trypan Blue Staining Cell Viability Assay Kit</td><td>Beyotime</td><td>C0011</td><td>Trypan Blue Staining</td></tr></tbody></table>

separation of immune cell subpopulations in peripheral blood samples from children with infectious mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;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.

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) are selected separately on a dot plot showing the CD3 PerCP-Cy5.5-A versus CD19 FITC-A. CD8+ T cells and CD4+ T cells are selected separately on a dot plot showing CD8 PE-A versus CD4 APC-Cy7-A from P3. CD16+/CD56+ NK cells are selected on a dot plot showing CD56/CD16 APC-A versus SSC-A from P4 (Figure 1C).
The representative results of four cell subpopulations sorted from the samples of patients with IM by the described method are shown in Figure 2A. We also performed cell sorting on samples from healthy EBV carriers and EBV-uninfected children as control groups to confirm the feasibility of this experiment. The representative results of cell subpopulations separated from a healthy EBV carrier&#39;s sample are shown in Figure 2B. The representative result of cell subpopulations sorted from the sample of EBV-uninfected children is shown in Figure 2C. As shown in Figure 2, P1 was gated to identify lymphocytes and doublet cells were excluded through P2; P3 was gated to select CD3+ T cells and P5 was gated to select CD19+ B cells; CD3+ CD8+ T cells (P6) and CD3+ CD4+ T cells (P7) were selected separately from P3; CD16+/CD56+ NK cells (P8) were selected from P4. Each of these subpopulations can be individually sorted and collected for downstream experiments. This system was used to analyze gene expression through RNA extraction and transcriptome sequencing.
As reported in Table 3, an increase in CD3+ CD8+ T cells was observed in patients with IM compared to both healthy EBV carriers and EBV-uninfected children (46.5 &#177; 4.0 vs. 27.0 &#177; 0.1 and 46.5 &#177; 4.0 vs. 24.7 &#177; 2.9, % CD3+ CD8+ T cells per total lymphocytes); Decreased proportions of CD3+ CD4+ T cells and CD19+ B cells were observed in patients with IM compared with healthy EBV carriers and EBV-uninfected children (13.4 &#177; 1.5 vs. 19.3 &#177; 1.5 and 13.4 &#177; 1.5 vs. 23.6 &#177; 3.2, % CD3+ CD4+ T cells per total lymphocytes; 1.4 &#177; 0.3 vs. 7.6 &#177; 0.7 and 1.4 &#177; 0.3 vs. 9.0 &#177; 1.5, % CD19+ B cells per total lymphocytes). Decreased proportions of CD16+/CD56+ NK cells were observed in patients with IM compared with EBV-uninfected children (7.5 &#177; 0.5 vs. 10.7 &#177; 0.4, % CD16+/CD56+ NK cells per total lymphocytes). These results validated the effectiveness of this sorting protocol and demonstrated that the proportions of lymphocyte subsets are different in patients with IM and EBV-uninfected children.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64212/64212fig01.jpg" /> 
Figure 1: Overall gating strategy used to sort immune cell subpopulations from PBMCs. (A) The PerCP-Cy5.5 filter was used to separate CD3+ T cells. CD8+ T cells and CD4+ T cells were selected separately on a dot plot showing CD8 PE-A versus CD4 APC-Cy7-A from P3. (B) The FITC filter was used to identify CD19+ B cells. (C) The APC filter was used to separate CD16+/CD56+ NK cells from P4. Abbreviations: PBMCs= peripheral blood mononuclear cells; FSC-A = forward scattering-area; SSC-A = side scattering-area; FSC-H = forward scattering-height; PerCP = peridinin-chrorophyll-protein; PE = phycoerythrin; APC = allophycocyanin; FITC = fluorescein isothiocyanate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64212/64212fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64212/64212fig02.jpg" /> 
Figure 2: Representative results of four cell subpopulations successfully isolated by the described method. (A) Representative cell sorting figures from the peripheral blood sample of patient with IM. (B) Representative cell sorting figures from the peripheral blood sample of healthy EBV carrier. (C) Representative cell sorting figures from the peripheral blood sample of EBV-uninfected children. P1, dot plot gate to identify lymphocytes; P2, dot plot gate to select single cells; P3, dot plot gate to select CD3+ T cells; P4, dot plot gate to identify CD3- CD19- lymphocytes; P5, dot plot gate to select CD19+ B cells; P6, dot plot gate to select CD3+ CD8+ T cells from P3; P7, dot plot gate to select CD3+ CD4+ T cells from P3; P8, dot plot gate to select CD16+/CD56+ NK cells from P4. Abbreviations: EBV = Epstein&#8211;Barr virus; IM = infectious mononucleosis; FSC-A = forward scattering-area; SSC-A = side scattering-area; FSC-H = forward scattering-height; PerCP = peridinin-chrorophyll-protein; PE = phycoerythrin; APC = allophycocyanin; FITC = fluorescein isothiocyanate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64212/64212fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody Target</td>
			<td>Conjugated Fluorophore</td>
			<td>Dosage</td>
			<td>Clone</td>
			<td>Isotype</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>PerCP/Cyanine5.5</td>
			<td>2 &#181;L</td>
			<td>SK7</td>
			<td>Mouse IgG1, &#954;</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>APC/Cyanine7</td>
			<td>2 &#181;L</td>
			<td>SK3</td>
			<td>Mouse IgG1, &#954;</td>
		</tr>
		<tr>
			<td>CD8</td>
			<td>PE</td>
			<td>2 &#181;L</td>
			<td>SK1</td>
			<td>Mouse IgG1, &#954;</td>
		</tr>
		<tr>
			<td>CD19</td>
			<td>FITC</td>
			<td>2 &#181;L</td>
			<td>HIB19</td>
			<td>Mouse IgG1, &#954;</td>
		</tr>
		<tr>
			<td>CD56</td>
			<td>APC</td>
			<td>2 &#181;L</td>
			<td>5.1H11</td>
			<td>Mouse IgG1, &#954;</td>
		</tr>
		<tr>
			<td>CD16</td>
			<td>APC</td>
			<td>2 &#181;L</td>
			<td>3G8</td>
			<td>Mouse IgG1, &#954;</td>
		</tr>
	</tbody>
</table>
Table 1: Antibodies used for flow cytometry. Abbreviations: PerCP = peridinin-chrorophyll-protein; PE = phycoerythrin; APC = allophycocyanin; FITC = fluorescein isothiocyanate.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="6">Compensation reference of flow cytometry (%)</td>
		</tr>
		<tr>
			<td></td>
			<td>PE</td>
			<td>APC</td>
			<td>APC-Cy7</td>
			<td>FITC</td>
			<td>PerCP-Cy5.5</td>
		</tr>
		<tr>
			<td>PE</td>
			<td>100</td>
			<td>0</td>
			<td>0</td>
			<td>0.8</td>
			<td>4.1</td>
		</tr>
		<tr>
			<td>APC</td>
			<td>0</td>
			<td>100</td>
			<td>30.1</td>
			<td>0</td>
			<td>0.9</td>
		</tr>
		<tr>
			<td>APC-Cy7</td>
			<td>0</td>
			<td>1.8</td>
			<td>100</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>FITC</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>100</td>
			<td>0</td>
		</tr>
		<tr>
			<td>PerCP-Cy5.5</td>
			<td>0</td>
			<td>1.8</td>
			<td>10</td>
			<td>0</td>
			<td>100</td>
		</tr>
	</tbody>
</table>
Table 2: Compensation reference of flow cytometry. Abbreviations: PerCP = peridinin-chrorophyll-protein; PE = phycoerythrin; APC = allophycocyanin; FITC = fluorescein isothiocyanate.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">Proportion of lymphocytes of different subpopulations in total sorted lymphocytes (%)</td>
		</tr>
		<tr>
			<td>lymphocytes subpopulation</td>
			<td>IM&#160;</td>
			<td>healthy EBV carriers</td>
			<td>EBV-uninfected children</td>
		</tr>
		<tr>
			<td>CD3+ T cells</td>
			<td>80.5 &#177; 1.8</td>
			<td>70.2 &#177; 2.3</td>
			<td>66.1 &#177; 2.1</td>
		</tr>
		<tr>
			<td>CD3+ CD8+&#160;T cells</td>
			<td>46.5 &#177; 4.0</td>
			<td>27.0 &#177; 0.1</td>
			<td>24.7 &#177; 2.9</td>
		</tr>
		<tr>
			<td>CD3+ CD4+&#160;T cells</td>
			<td>13.4 &#177; 1.5</td>
			<td>19.3 &#177; 1.5</td>
			<td>23.6 &#177; 3.2</td>
		</tr>
		<tr>
			<td>&#160;CD16+/CD56+&#160;NK cells</td>
			<td>7.5 &#177; 0.5</td>
			<td>2.2 &#177; 0.1</td>
			<td>10.7 &#177; 0.4</td>
		</tr>
		<tr>
			<td>CD3- CD19+ B cells</td>
			<td>1.4 &#177; 0.3</td>
			<td>7.6 &#177; 0.7</td>
			<td>9.0 &#177; 1.5</td>
		</tr>
	</tbody>
</table>
Table 3: The proportion of sorted lymphocytes of different groups. Abbreviations: EBV = Epstein&#8211;Barr virus; IM = infectious mononucleosis; NK = natural killer.

Watch this Scientific Journal Video about Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis at JoVE.com

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

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&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

separation-immune-cell-subpopulations-peripheral-blood-samples-from

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

2.3K Views.  Capital Medical University, National Center for Children's Health. Deze methode zal de karakterisering van verschillende immuuncellen in dezelfde ziektetoestanden van personen met IM mogelijk maken, wat ons begrip van het mechanisme van EBV-infectie zal vergroten. De procedure wordt gedemonstreerd door Linlin Zhang, een arts van ons laboratorium. Neem om te beginnen de PBMC's die eerder zijn geïsoleerd in een microcentrifugebuis van 1,5 milliliter en draai deze gedurende 10 minuten op 300 G bij kamertemperatuur.Gooi het supernatant weg en suspensieer...

Video: Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Biology

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides (MF) and S&#233;zary syndrome (SS) represent the most common subtypes. Despite a number of studies on characterizing gene expression, genetic alterations, and epigenetic abnormalities of CTCL, the molecular pathogenesis of MF/SS remains unclear. MF refers to the more common CTCL with a skin-predominance, and is usually limited to skin, whereas&#160;SS is an aggressive leukemic variant of CTCL with widespread skin involvement and is characterized by neoplastic distribution mainly involving blood, skin, and lymph node. Focusing on clinical practice, the identification of gene expression biomarkers has&#160;enormous potential to improve diagnosis and treatment of MF/SS. Indeed, recent transcriptomic studies have identified potential diagnostic biomarkers from differences in gene expression between normal and malignant T cells, which may improve our understanding of SS biology, and reveal potential therapeutic targets. This manuscript describes a detailed reproducible protocol for the isolation of peripheral blood mononuclear cells from fresh whole blood from patients diagnosed with SS, selection of CD4+ memory T cells (CD4+CD45RO+ T cells), chemical stimulation, and preparation of RNA suitable for transcriptomic profiling to discover novel prognostic molecular markers to gain additional insight in disease etiology. The stimulation using chemical agonist to activate nuclear regulation provides more specific assessment for pathways important in the dynamic transcription regulation and gene expression and eliminates confounding defects that may arise from upstream signaling defects arising from TCR antigen loss at the cell membrane. The data obtained from comparison of transcriptome of unstimulated to stimulated SS T cells unmasks functional regulatory gene expression defects not evident from analysis of quiescent unstimulated cells. Furthermore, the method outlined from this approach can be adapted for studying T cell gene expression defects in other T cell immune diseases.

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with S&#233;zary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

Cancer Research

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

High-Throughput PBMC Isolation Using Immunomagnetic Beads

Isolating Human Peripheral Blood Mononuclear Cells from Buffy Coats via High Throughput Immunomagnetic Bead Separation

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in long-term storage, making them an ideal cell type for many downstream research purposes, including flow cytometry, immunoassays, and genome sequencing. Typically, PBMCs are isolated via&#160;density gradient centrifugation, however, it is a low-throughput workflow that is difficult and costly to scale. This article presents a high-throughput workflow using a magnetic bead-based PBMC isolation method that is quick to implement. Total cell concentration, viability, and population distribution with PBMCs obtained using density gradient isolation were compared, and cell viability and proportion of cell types were comparable for both techniques. Isolated PBMCs demonstrated over 70% viability up to 9 days after blood collection, although yield decreased by half after 5 days compared to PBMCs processed within 24 h of collection. In summary, this article describes a PBMC protocol that utilizes a bead-based approach to adapt to a high throughput workflow and demonstrates that both manual and automated bead-based methods can increase processing capacity and provide flexibility for various budgets.

Author Spotlight: Advancements in PBMC Isolation &#8211; Enhancing Throughput and Consistency in Research

This protocol details a high-throughput automation-compatible method to isolate human peripheral blood mononuclear cells for biobanking and other purposes.

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in ...

Isolation of Monocytes from Whole Blood by Immunomagnetic Negative Selection

Source: Plaisance-Bonstaff, K., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59967">Isolation, Transfection, and Culture of Primary Human Monocytes.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the isolation of monocytes from whole human blood by immunomagnetic negative separation. The isolated monocytes can be used for further downstream analysis.

Source: Plaisance-Bonstaff, K., et al. Isolation, Transfection, and Culture of Primary Human Monocytes. J. Vis. Exp. (2019)
In this video, we demonstrate ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Isolation and Culture

Isolation of Peripheral Blood Mononuclear Cells from Human Buffy Coats

Source: Mily, A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61807">Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection.</a> J. Vis. Exp. (2020)
In this video, we demonstrate the isolation of peripheral blood mononuclear cells, PBMCs, containing monocytes and lymphocytes from a human buffy coat by density gradient centrifugation. The isolated PBMCs can be used for further analysis.

Source: Mily, A., et al. Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis ...

Isolation and Purification of B Cells from Human Peripheral Blood

Source: Tzeng, S. J. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54582">The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity.</a> J. Vis. Exp. (2016)
In this video, we demonstrate the isolation of B cells from human peripheral blood using biotinylated antibodies specific for non-B cells and streptavidin-conjugated magnetic microbeads.

Source: Tzeng, S. J. The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

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