Name: separation of immune cell subpopulations in peripheral blood samples from children with infectious mononucleosis
Uploaded: 2022-09-07T13:00:00
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...

<ol>
	<li>Kwok, H., Chiang, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+conventional+to+next+generation+sequencing+of+Epstein-Barr+virus+genomes.">From conventional to next generation sequencing of Epstein-Barr virus genomes.</a> Viruses. 8 (3), 60 (2016).</li><li>Bu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+with+components+of+the+viral+fusion+apparatus+elicits+antibodies+that+neutralize+Epstein-Barr+virus+in+B+cells+and+epithelial+cells.">Immunization with components of the viral fusion apparatus elicits antibodies that neutralize Epstein-Barr virus in B cells and epithelial cells.</a> Immunity. 50 (5), 1305-1316 (2019).</li><li>Balfour, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral,+virology,+and+immunologic+factors+associated+with+acquisition+and+severity+of+primary+Epstein-Barr+virus+infection+in+university+students.">Behavioral, virology, and immunologic factors associated with acquisition and severity of primary Epstein-Barr virus infection in university students.</a> Journal of Infectious Diseases. 207 (1), 80-88 (2013).</li><li>Luzuriaga, K., Sullivan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectious+mononucleosis.">Infectious mononucleosis.</a> New England Journal of Medicine. 362 (21), 1993-2000 (2010).</li><li>Fujiwara, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+research+on+chronic+active+Epstein-Barr+virus+infection+in+Japan.">Current research on chronic active Epstein-Barr virus infection in Japan.</a> Pediatrics International. 56 (2), 159-166 (2014).</li><li>Tsao, S. W., Tsang, C. M., Lo, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus+infection+and+nasopharyngeal+carcinoma.">Epstein-Barr virus infection and nasopharyngeal carcinoma.</a> Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences. 372 (1732), 20160270 (2017).</li><li>Zhong, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+transcriptome+profiling+reveals+major+cell+types+in+the+cellular+immune+response+against+acute+and+chronic+active+Epstein-Barr+virus+infection.">Whole transcriptome profiling reveals major cell types in the cellular immune response against acute and chronic active Epstein-Barr virus infection.</a> Scientific Reports. 7 (1), 17775 (2017).</li><li>Fedyanina, O. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+and+clinical+significance+of+atypical+mononuclear+cells+in+infectious+mononucleosis+caused+by+the+Epstein-Barr+virus+in+children.">The nature and clinical significance of atypical mononuclear cells in infectious mononucleosis caused by the Epstein-Barr virus in children.</a> Journal of Infectious Diseases. 223 (10), 1699-1706 (2021).</li><li>Al Tabaa, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-cell+polyclonal+activation+and+Epstein-Barr+viral+abortive+lytic+cycle+are+two+key+features+in+acute+infectious+mononucleosis.">B-cell polyclonal activation and Epstein-Barr viral abortive lytic cycle are two key features in acute infectious mononucleosis.</a> Journal of Clinical Virology. 52 (1), 33-37 (2011).</li><li>M, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+Killer+cell+transcriptome+during+primary+EBV+infection+and+EBV+associated+Hodgkin+Lymphoma+in+children-A+preliminary+observation.">Natural Killer cell transcriptome during primary EBV infection and EBV associated Hodgkin Lymphoma in children-A preliminary observation.</a> Immunobiology. 225 (3), 151907 (2020).</li><li>Greenough, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+expression+signature+that+correlates+with+CD8++T+cell+expansion+in+acute+EBV+infection.">A gene expression signature that correlates with CD8+ T cell expansion in acute EBV infection.</a> Journal of Immunology. 195 (9), 4185-4197 (2015).</li><li>Xia, Y., Gao, Y., Wang, B., Zhang, H., Zhang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+the+method+of+cell+separation+from+bile+of+patients+with+cholangiocarcinoma+for+flow+cytometry.">Optimizing the method of cell separation from bile of patients with cholangiocarcinoma for flow cytometry.</a> Gastroenterology Research and Practice. , 5436961 (2019).</li><li>Maecker, H. T., Trotter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+controls,+instrument+setup,+and+the+determination+of+positivity.">Flow cytometry controls, instrument setup, and the determination of positivity.</a> Cytometry Part A. 69 (9), 1037-1042 (2006).</li><li>Byford, E., Carr, M., Pinon, L., Ahearne, M. J., Wagner, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CD4++T-cells+and+analysis+of+circulating+T-follicular+helper+(cTfh)+cell+subsets+from+peripheral+blood+using+6-color+flow+cytometry.">Isolation of CD4+ T-cells and analysis of circulating T-follicular helper (cTfh) cell subsets from peripheral blood using 6-color flow cytometry.</a> Journal of Visualized Experiments. (143), e58431 (2019).</li><li>Djaoud, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+alternate+strategies+for+innate+immunity+to+Epstein-Barr+virus:+One+using+NK+cells+and+the+other+NK+cells+and+gammadelta+T+cells.">Two alternate strategies for innate immunity to Epstein-Barr virus: One using NK cells and the other NK cells and gammadelta T cells.</a> Journal of Experimental Medicine. 214 (6), 1827-1841 (2017).</li><li>Nakid-Cordero, C., Baron, M., Guihot, A., Vieillard, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+killer+cells+in+post-transplant+lymphoproliferative+disorders.">Natural killer cells in post-transplant lymphoproliferative disorders.</a> Cancers. 13 (8), 1836 (2021).</li><li>Forrest, C., Hislop, A. D., Rickinson, A. B., Zuo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome-wide+analysis+of+CD8++T+cell+responses+to+EBV+reveals+differences+between+primary+and+persistent+infection.">Proteome-wide analysis of CD8+ T cell responses to EBV reveals differences between primary and persistent infection.</a> PLoS Pathogens. 14 (9), 1007110 (2018).</li><li>Zamai, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+Synergy+of+NKp46+and+co-activating+signals+in+various+NK+cell+subpopulations:+paving+the+way+for+more+successful+NK-cell-based+immunotherapy.">Understanding the Synergy of NKp46 and co-activating signals in various NK cell subpopulations: paving the way for more successful NK-cell-based immunotherapy.</a> Cells. 9 (3), 753 (2020).</li><li>Lam, J. K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+CD4++and+CD8++polyfunctional+T+cell+responses+against+immunodominant+lytic+and+latent+EBV+antigens+in+children+with+primary+EBV+infection.">Emergence of CD4+ and CD8+ polyfunctional T cell responses against immunodominant lytic and latent EBV antigens in children with primary EBV infection.</a> Frontiers In Microbiology. 9, 416 (2018).</li><li>Choi, I. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+by+the+Epstein-Barr+virus+LMP1+protein+induces+potent+cytotoxic+CD4(+)+and+CD8(+)+T+cell+responses.">Signaling by the Epstein-Barr virus LMP1 protein induces potent cytotoxic CD4(+) and CD8(+) T cell responses.</a> Proceedings of the National Academy of Sciences. 115 (4), 686-695 (2018).</li></ol>

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...

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.

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	<tbody>
		<tr>
			<td>APC anti-human CD16</td>
			<td>Biolegend</td>
			<td>302012</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>APC anti-human CD56 (NCAM)</td>
			<td>Biolegend</td>
			<td>362504</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>APC/Cyanine7 anti-human CD4</td>
			<td>Biolegend</td>
			<td>344616</td>
			<td>Fluorescent antibody&#160;</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&#160;flow&#160;cell&#160;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 (&#8805;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>&#160;367862&#160;</td>
			<td>EDTA anticoagulant tubes</td>
		</tr>
		<tr>
			<td>FITC anti-human CD19</td>
			<td>Biolegend</td>
			<td>302206</td>
			<td>Fluorescent antibody&#160;</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>&#160;High-speed&#160;centrifuge</td>
			<td>Sigma</td>
			<td>&#160;3K15</td>
			<td>Cell centrifugation for 15 mL centrifuge tube</td>
		</tr>
		<tr>
			<td>&#160;High-speed&#160;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&#160;Separation&#160;columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>Separation&#160;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&#160;</td>
		</tr>
		<tr>
			<td>PerCP/Cyanine5.5 anti-human CD3</td>
			<td>Biolegend</td>
			<td>344808</td>
			<td>Fluorescent antibody&#160;</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) ar...

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 ...

This method will enable the characterization of different immune cells in the same states of disease of individuals with IM, which will expand our understanding of the mechanism of EBV infection. Demonstrating the procedure will be Linlin Zhang, a doctor from our laboratory. To begin, take the PBMCs isolated previously in a 1.5 milliliter microcentrifuge tube, and spin it at 300 G for 10 minutes at room temperature.Discard the supernatant, and re-suspend the cells with 80 microliters of the prepared buffer. Next, add 20 microliters of CD 14 microbeads to the cell suspension, mixed well by pipetting, and incubate the tube for 15 minutes on ice. Then wash the PBMCs using one milliliter of buffer and centrifuge at 300 G for 10 minutes at room temperature.Discard the entire supernatant, and re-suspend the cells with 500 microliters of the buffer. For magnetic separation, place a column on the magnetic bead separator, and prime the column by washing it with three milliliters of the buffer. Next, add the cell suspension to the column.Allow it to pass through, and collect the unlabeled cells into a 15 milliliter centrifuge tube. Wash the column three times with three milliliters of the buffer, and collect the entire effluent in a 15 milliliter centrifuge tube. Now, place the column in a new 15 milliliters centrifuge tube, and add five milliliters of buffer to the column.Push the plunger firmly into the column to flush the magnetically labeled cells into the tube. Centrifuge the magnetically labeled cells at 300 G for five minutes. After removing the supernatant, re-suspend the cells in 500 microliters of PBS, and transfer the cell suspension to a 1.5 milliliter microcentrifuge tube for subsequent transcriptome sequencing.Pellet the collected unlabeled cells by centrifuging at 300 G for five minutes, and re-suspend the cells in 100 microliters of PBS in a 1.5 milliliter microcentrifuge tube. Then, add two microliters of each labeled antibody to the cell suspension, and incubate on ice for 30 minutes, protected from light. Next, aliquot 100 microliters of the PBMC cell suspension in 1.5 milliliter microcentrifuge tubes to set up a negative control sample CD 3, CD 4, CD 8, and CD 19, single staining samples, and a CD 56, or CD 16 staining sample.Then add two microliters of the corresponding fluorescently labeled antibody to each tube, and incubate on ice for 30 minutes, protected from light. After incubation, wash all cells with PBS as demonstrated previously, and re-suspend in 500 microliters of PBS. Open the cell sorting system, and run the power on program.Then, install the 85 micrometer nozzle, and open the sorting stream. Set the sorting voltage to 4, 500 volts, and the frequency to 47. Set up the gap to 8 in the break-off window, and turn on the sweet spot automatic sorting mode to determine the droplet amplitude value automatically.Finally, adjust the droplet delay to 30.31 in the side stream window. Using a forward versus side scatter dot plot, draw the polygon gate P1 to identify the intact lymphocyte population. Next, using a forward scatter area versus height dot plot, draw the polygon gate P2 to identify the single cells, and exclude doublets.Then using a CD 19 FITC area versus CD 3 per CPCY 5.5 area dot plot, Draw the rectangular gate P3 to select CD 3 positive T-cells, and CD 19 positive B-cells. Next, using a CD 4 APCCY 7 area versus CD 8 PE area dot plot, draw a rectangular gate to select CD 4, and CD 8 positive T-cells. Finally, using a side scatter area versus CD 56 or CD 16 APC area dot plot, draw a rectangular gate to select CD 56, or CD 16 positive natural killer cells.For the negative control tube, click load in the acquisition dashboard, and select the cytometer settings in the software. In the inspector window, click the parameters tab, and adjust the voltages of forward scatter, side scatter, and different fluorescent dyes. After loading the single stained tubes into the cytometer, click load in the acquisition dashboard, then click the compensation tab to adjust the compensation, as described in the text.Vortex the cell suspension gently, before loading the tube into the cytometer. Add 200 microliters of FBS to four collection flow tubes, and vortex the tubes. Place them in the cytometer collection chamber.Collect the different types of cells separately in the four flow tubes. Spin down the separated cells at 300 G for five minutes, and add 200 microliters of RNA isolation reagent to the cell palette for transcriptome sequencing. In the present study, immune cells subpopulations from peripheral blood of different samples were sorted out efficiently by combining the techniques of immunomagnetic beads, and fluorescence activated cell sorting.Importantly in this protocol, the cell sorting step has been optimized to improve cell viability. The sorted immune cells showed an increased proportion of CD 3, CD 8 positive T-cells in patients with infectious mononucleosis compared to both the healthy Epstein-Barr virus carriers, and uninfected samples. In contrast, decreased proportions of CD 3, CD 4 positive T-cells, and CD 19 positive B-cells were observed in patients with infectious mononucleosis compared with healthy EBV carriers, and EBV uninfected children.Additionally, decreased proportions of CD 56, or CD 16 positive natural killer cells were observed in patients with infectious mononucleosis compared with EBV uninfected children. Maintaining cell viability is vital for subsequent experiment. Keeping cells on ice, or in the refrigerator during the sorting process is beneficial for cell viability.

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Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

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 ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...