This study was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases, &#60;https://www.niams.nih.gov/&#62;, award number P30-AR053503; The Stabler Foundation,&#160;www.stablerfoundation.org; National Institute of Allergy and Infectious Disease, www.niaid.nih.gov, T32AI007247; Nina Ireland Program for Lung Health (NIPLH), https://pulmonary.ucsf.edu/ireland/.

All studies of human materials were approved by the Johns Hopkins Institutional Review Board under the Health Insurance Portability and Accountability Act. Patient and control samples were de-identified. PBMCs and blood from healthy controls were obtained by informed consent.
NOTE: This protocol has been tested on freshly or frozen isolated peripheral blood cells and whole blood.
1. Cell Preparation
<ol>
	<li>Isolation of peripheral blood ...

<ol>
	<li>Bendall, S. C., Nolan, G. P., Roederer, M., Chattopadhyay, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+deep+profiler's+guide+to+cytometry.">A deep profiler's guide to cytometry.</a> Trends in Immunology. 33 (7), 323-332 (2012).</li><li>Boin, F., 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=Flow+cytometric+discrimination+of+seven+lineage+markers+by+using+two+fluorochromes.">Flow cytometric discrimination of seven lineage markers by using two fluorochromes.</a> PLoS ONE. 12 (11), (2017).</li><li>Zola, 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=.">.</a> Leukocyte and Stromal Cell Molecules: The CD Markers. , (2007).</li><li>Lambert, C., Genin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD3+bright+lymphocyte+population+reveal+&#947;&#948;+T+cells.">CD3 bright lymphocyte population reveal &#947;&#948; T cells.</a> Cytometry Part B: Clinical Cytometry. 61 (1), 45-53 (2004).</li><li>Ginaldi, 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=Differential+expression+of+T+cell+antigens+in+normal+peripheral+blood+lymphocytes:+a+quantitative+analysis+by+flow+cytometry.">Differential expression of T cell antigens in normal peripheral blood lymphocytes: a quantitative analysis by flow cytometry.</a> Journal of Clinical Pathology. 49 (7), 539-544 (1996).</li><li>Park, J., Han, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-color+Multitarget+Flow+Cytometry+Using+Monoclonal+Antibodies+Labeled+with+Different+Intensities+of+the+Same+Fluorochrome.">Single-color Multitarget Flow Cytometry Using Monoclonal Antibodies Labeled with Different Intensities of the Same Fluorochrome.</a> Annals of Laboratory Medicine. 32 (3), 171-176 (2012).</li><li>Mansour, I., 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=Triple+labeling+with+two-color+immunoflorescence+using+one+light+source:+A+useful+approach+for+the+analysis+of+cells+positive+for+one+label+and+negative+for+the+other+two.">Triple labeling with two-color immunoflorescence using one light source: A useful approach for the analysis of cells positive for one label and negative for the other two.</a> Cytometry. 11 (5), 636-641 (1990).</li><li>Bocsi, J., Melzer, S., D&#228;hnert, I., T&#225;rnok, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OMIP-023:+10-Color,+13+antibody+panel+for+in-depth+phenotyping+of+human+peripheral+blood+leukocytes.">OMIP-023: 10-Color, 13 antibody panel for in-depth phenotyping of human peripheral blood leukocytes.</a> Cytometry Part A. 85 (9), 781-784 (2014).</li><li>Van Dongen, J. J. M., 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=EuroFlow+antibody+panels+for+standardized+n-dimensional+flow+cytometric+immunophenotyping+of+normal,+reactive+and+malignant+leukocytes.">EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes.</a> Leukemia. 26 (9), 1908-1975 (2012).</li><li>Bradford, J. A., Buller, G., Suter, M., Ignatius, M., Beechem, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-intensity+multiplexing:+Simultaneous+seven-marker,+two-color+immunophenotyping+using+flow+cytometry.">Fluorescence-intensity multiplexing: Simultaneous seven-marker, two-color immunophenotyping using flow cytometry.</a> Cytometry Part A. 61 (2), 142-152 (2004).</li><li>R&#252;hle, P. F., Fietkau, R., Gaipl, U. S., Frey, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Modular+Assay+for+Detailed+Immunophenotyping+of+Peripheral+Human+Whole+Blood+Samples+by+Multicolor+Flow+Cytometry.">Development of a Modular Assay for Detailed Immunophenotyping of Peripheral Human Whole Blood Samples by Multicolor Flow Cytometry.</a> International Journal of Molecular Sciences. 17 (8), (2016).</li><li>H&#248;nge, B. L., Petersen, M. S., Olesen, R., M&#248;ller, B. K., Erikstrup, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+recovery+of+frozen+human+peripheral+blood+mononuclear+cells+for+flow+cytometry.">Optimizing recovery of frozen human peripheral blood mononuclear cells for flow cytometry.</a> PLOS ONE. 12 (11), e0187440 (2017).</li><li>Roederer, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FACS+analysis+of+lymphocytes.">FACS analysis of lymphocytes.</a> Handbook of Experimental Immunology. 49, (1997).</li><li>Srivastava, P., Sladek, T. L., Goodman, M. N., Jacobberger, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptavidin-based+quantitative+staining+of+intracellular+antigens+for+flow+cytometric+analysis.">Streptavidin-based quantitative staining of intracellular antigens for flow cytometric analysis.</a> Cytometry. 13 (7), 711-721 (1992).</li><li>Maecker, H. T., 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=Selecting+fluorochrome+conjugates+for+maximum+sensitivity.">Selecting fluorochrome conjugates for maximum sensitivity.</a> Cytometry Part A. , (2004).</li><li>Noonan, K. 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=Adoptive+transfer+of+activated+marrow-infiltrating+lymphocytes+induces+measurable+antitumor+immunity+in+the+bone+marrow+in+multiple+myeloma.">Adoptive transfer of activated marrow-infiltrating lymphocytes induces measurable antitumor immunity in the bone marrow in multiple myeloma.</a> Science Translational Medicine. 7 (288), (2015).</li><li>Mahnke, Y. D., Beddall, M. H., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OMIP-017:+Human+CD4++helper+T-cell+subsets+including+follicular+helper+cells.">OMIP-017: Human CD4+ helper T-cell subsets including follicular helper cells.</a> Cytometry Part A. 83 (5), 439-440 (2013).</li><li>Bonecchi, R., 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=Differential+Expression+of+Chemokine+Receptors+and+Chemotactic+Responsiveness+of+Type+1+T+Helper+Cells+(Th1s)+and+Th2s.">Differential Expression of Chemokine Receptors and Chemotactic Responsiveness of Type 1 T Helper Cells (Th1s) and Th2s.</a> The Journal of Experimental Medicine. 187 (1), 129-134 (1998).</li><li>Acosta-Rodriguez, E. V., 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=Surface+phenotype+and+antigenic+specificity+of+human+interleukin+17-producing+T+helper+memory+cells.">Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells.</a> Nature Immunology. 8 (6), 639-646 (2007).</li><li>Rivino, 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=Chemokine+Receptor+Expression+Identifies+Pre-T+Helper+(Th)1,+Pre-Th2,+and+Nonpolarized+Cells+among+Human+CD4++Central+Memory+T+Cells.">Chemokine Receptor Expression Identifies Pre-T Helper (Th)1, Pre-Th2, and Nonpolarized Cells among Human CD4+ Central Memory T Cells.</a> The Journal of Experimental Medicine. 200 (6), 725-735 (2004).</li><li>Ye, Z. -. J., 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=Differentiation+and+recruitment+of+Th9+cells+stimulated+by+pleural+mesothelial+cells+in+human+Mycobacterium+tuberculosis+infection.">Differentiation and recruitment of Th9 cells stimulated by pleural mesothelial cells in human Mycobacterium tuberculosis infection.</a> PloS One. 7 (2), e31710 (2012).</li><li>Speiser, D. E., 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=Human+CD8++T+cells+expressing+HLA-DR+and+CD28+show+telomerase+activity+and+are+distinct+from+cytolytic+effector+T+cells.">Human CD8+ T cells expressing HLA-DR and CD28 show telomerase activity and are distinct from cytolytic effector T cells.</a> European Journal of Immunology. 31 (2), 459-466 (2001).</li><li>Caruso, 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=Flow+cytometric+analysis+of+activation+markers+on+stimulated+T+cells+and+their+correlation+with+cell+proliferation.">Flow cytometric analysis of activation markers on stimulated T cells and their correlation with cell proliferation.</a> Cytometry. 27 (1), 71-76 (1997).</li><li>Brenchley, J. M., 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=Expression+of+CD57+defines+replicative+senescence+and+antigen-induced+apoptotic+death+of+CD8++T+cells.">Expression of CD57 defines replicative senescence and antigen-induced apoptotic death of CD8+ T cells.</a> Blood. 101 (7), 2711-2720 (2003).</li><li>Palmer, B. E., Blyveis, N., Fontenot, A. P., Wilson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+Phenotypic+Characterization+of+CD57+CD4++T+Cells+and+Their+Association+with+HIV-1-Induced+T+Cell+Dysfunction.">Functional and Phenotypic Characterization of CD57+CD4+ T Cells and Their Association with HIV-1-Induced T Cell Dysfunction.</a> The Journal of Immunology. 175 (12), 8415-8423 (2005).</li><li>Focosi, D., Bestagno, M., Burrone, O., Petrini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD57++T+lymphocytes+and+functional+immune+deficiency.">CD57+ T lymphocytes and functional immune deficiency.</a> Journal of Leukocyte Biology. 87 (1), 107-116 (2010).</li></ol>

The protocol presented here has been shown to be quite flexible and insensitive to changes in staining buffer, temperature and peripheral blood cell preparation due to the high expression of lineage markers on the cell surface. The most critical step for obtaining high-quality, reproducible data is antibody titration. Of note, since the titration of antibodies should always be performed during the setup of a flow cytometric panel, this step does not add extra bench-time to our two-fluorochrome approach. Titration of anti...

Flow cytometry is a technique that was developed to analyze multiple parameters on single particles at a rate of several thousand of events per second1. Examples of specimens analyzed by flow cytometry include, but are not limited to, cells, beads, bacteria, vesicles and chromosomes. A fluidic system directs particles at the interrogation point where each particle intersects its path with one or more lasers, and multiple parameters are recorded for further analysis. Forward and side scatters, generated by scattering of the pure laser light, are used to identify the target population and retrieve information about the relative size and internal complexity/granularity of particles, respectively. All the other parameters, that account for most of data in a flow cytometric analysis, are derived by fluorochrome-labeled probes that recognize and bind to specific targets on the particles of interest.
Flow cytometry is a primary tool for immunological studies to identify and characterize cell populations. To dissect the complexity of the immune system, multicolor panels are constantly evolving to expand the number of markers simultaneously recorded for deep immunophenotyping of cell populations1. This is leading to the development of more capable instruments and fluorochromes, with recent high-end flow cytometers exceeding 20 fluorescent parameters. This results in complex study design due to fluorochrome spectral overlap and in higher costs associated with custom antibody labelling and skilled operators. In several instances, complexity and costs are reduced by using separate panels of markers for different cell populations. This approach, however, is error prone, reduces the information in each panel, and can be difficult to apply to samples with limited number of cells. Moreover, increasing the number of markers precludes deep immunophenotyping on instruments with fewer fluorescent parameters. We previously developed a staining protocol to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood mononuclear cells (PBMCs) by combining seven lineage markers using only two fluorochromes instead of the seven required using the traditional &#34;one fluorochrome-one marker&#34; approach (www.hcdm.org)2,3.&#160;Our initial report explored and validated the notion of combining seven markers in two fluorochromes for deep immunophenotyping. In this report, we present a step-by step protocol to isolate and stain peripheral blood cells, focusing on the practical aspect and troubleshooting steps to achieve a successful staining.&#160;
This protocol is based on the observation that lineage markers have a constant expression on the cell surface and that each cell population has an exclusive combination of lineage markers. In PBMCs, CD3 expression subdivides immune cells into two main categories: CD3-positive T lymphocytes and CD3-negative cells. Within the CD3 positive subgroup, CD4+, CD8+ and &#947;&#948; T cells can be separated using antibodies that solely target CD4, CD8 and the &#947;&#948; receptor. In a comparable way, within the CD3 negative subgroup, B cells, NK cells and monocytes can be uniquely identified using antibodies against CD19, CD56 and CD14, respectively. In a standard one fluorochrome-one marker approach, anti-CD3, -CD4, -CD8, -CD14, -CD19, -CD56 and -TCR &#947;&#948; antibodies are detected with seven different fluorochromes. Our approach combines anti-CD3, -CD56, and -TCR &#947;&#948; antibodies in one fluorochrome (labeled for convenience fluorochrome A) and anti-CD4, -CD8, -CD14 and -CD19 antibodies in a different fluorochrome (fluorochrome B). This is possible by a combination of antibody titration and differential antigen expression. Both CD4+ and CD8+ T cells are positive for the anti-CD3 antibody in fluorochrome A, but they can be separated in fluorochrome B maximizing the expression of the CD8 signal while placing, with an ad hoc titration, the CD4 signal in between the CD8 and the CD3 positive-CD4/CD8 double negative cells. &#947;&#948; T cells expresses higher level of CD3 than CD4 and CD8, and therefore they can be identified as CD3 high4. This signal is further boosted by labeling &#947;&#948; T cells in fluorochrome A with an anti-TCR &#947;&#948; antibodies, thus improving separation between CD3 low T cells and CD3 high &#947;&#948; T cells. B cells can be identified as CD3- in fluorochrome A and CD19+ in fluorochrome B. To separate CD3 negative NK cells from B cells, an anti-CD56 antibody was used in fluorochrome A as the anti-CD3. This is possible because CD56 expressed on NK cell at a much lower level than CD3 on T cells5. Finally, monocytes can be identified via a combination of forward-side scatter properties and expression of CD14 in fluorochrome B.
The idea of combining up to four markers using two fluorochromes has been already successfully attempted before6,7,8, and has been used in a clinical protocol to identify malignant lymphocytic populations9. A previous report also combined seven markers (with different specificity from the markers than we used in our protocol) using two fluorochromes, but this approach relied on a complex labelling of each antibody with varying amount of fluorochrome10. This is in contrast to our method which uses commercially available antibodies and can be adapted to the instrument configuration and can take advantage of the new generation of polymer fluorochromes.
The overall goal of this methodology is to expand the optical limits of most flow cytometers allowing for the recording of five additional markers to interrogate complex cell populations. As a consequence, advanced immunological analysis can be performed on affordable 6-10 fluorochrome flow cytometers, and 2-3 fluorochrome field instruments can achieve remarkable results in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis and to create modular flow cytometric panels targeting several lineages at the same time11. This can potentially reduce the number of panels used in modular immunophenotyping flow cytometry and reduce costs, errors and handling time. This approach is also very well suited in the case of clinical samples with limited number of cells.

<table>
	<tbody>
		<tr>
			<td>CD3</td>
			<td>BD Biosciences</td>
			<td>562426</td>
			<td>Antibody for staining 
			RRID: AB_11152082</td>
		</tr>
		<tr>
			<td>CD56</td>
			<td>BD Biosciences</td>
			<td>562751</td>
			<td>Antibody for staining 
			RRID: AB_2732054</td>
		</tr>
		<tr>
			<td>TCRgd</td>
			<td>BD Biosciences</td>
			<td>331217</td>
			<td>Antibody for staining 
			RRID: AB_2562316</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>BD Biosciences</td>
			<td>555347</td>
			<td>Antibody for staining 
			RRID: AB_395752</td>
		</tr>
		<tr>
			<td>CD8</td>
			<td>BD Biosciences</td>
			<td>555367</td>
			<td>Antibody for staining 
			RRID: AB_395770</td>
		</tr>
		<tr>
			<td>CD14</td>
			<td>BD Biosciences</td>
			<td>555398</td>
			<td>Antibody for staining 
			RRID: AB_395799</td>
		</tr>
		<tr>
			<td>CD19</td>
			<td>BD Biosciences</td>
			<td>555413</td>
			<td>Antibody for staining 
			RRID: AB_395813</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>BD Biosciences</td>
			<td>555335</td>
			<td>Antibody for staining 
			RRID: AB_398591</td>
		</tr>
		<tr>
			<td>CD56</td>
			<td>BD Biosciences</td>
			<td>555518</td>
			<td>Antibody for staining 
			RRID: AB_398601</td>
		</tr>
		<tr>
			<td>TCRgd</td>
			<td>BD Biosciences</td>
			<td>331211</td>
			<td>Antibody for staining 
			RRID: AB_1089215</td>
		</tr>
		<tr>
			<td>CCR7</td>
			<td>Biolegend</td>
			<td>353217</td>
			<td>Antibody for staining 
			RRID: AB_10913812</td>
		</tr>
		<tr>
			<td>CD45RA</td>
			<td>BD Biosciences</td>
			<td>560674</td>
			<td>Antibody for staining 
			RRID: AB_1727497</td>
		</tr>
		<tr>
			<td>CCR4</td>
			<td>BD Biosciences</td>
			<td>561034</td>
			<td>Antibody for staining 
			RRID: AB_10563066</td>
		</tr>
		<tr>
			<td>CCR6</td>
			<td>BD Biosciences</td>
			<td>353419</td>
			<td>Antibody for staining 
			RRID: AB_11124539</td>
		</tr>
		<tr>
			<td>CXCR3</td>
			<td>BD Biosciences</td>
			<td>561730</td>
			<td>Antibody for staining 
			RRID: AB_10894207</td>
		</tr>
		<tr>
			<td>CD57</td>
			<td>BD Biosciences</td>
			<td>562488</td>
			<td>Antibody for staining 
			RRID: AB_2737625</td>
		</tr>
		<tr>
			<td>HLA-DR</td>
			<td>BD Biosciences</td>
			<td>563083</td>
			<td>Antibody for staining 
			RRID: AB_2737994</td>
		</tr>
		<tr>
			<td>CD16</td>
			<td>BD Biosciences</td>
			<td>563784</td>
			<td>Antibody for staining 
			RRID: AB_2744293</td>
		</tr>
		<tr>
			<td>Dead cells</td>
			<td>Life technologies</td>
			<td>L-23105</td>
			<td>Live/dead discrimination</td>
		</tr>
		<tr>
			<td>Falcon 5 ml round-bottom polystyrene test tube with cell strainer snap cap</td>
			<td>BD Bioscience</td>
			<td>352235</td>
			<td>to filter cell suspension before passing though the flow cytometer</td>
		</tr>
		<tr>
			<td>Falcon 5 ml round-bottom polystyrene test tube</td>
			<td>BD Bioscience</td>
			<td>352001</td>
			<td>to stain whole blood</td>
		</tr>
		<tr>
			<td>Recovery Cell Culture Freezing Medium</td>
			<td>Thermo fisher</td>
			<td>12648010</td>
			<td>Freezing cells</td>
		</tr>
		<tr>
			<td>96-well V-bottom plate</td>
			<td>Thermo fisher</td>
			<td>249570</td>
			<td>plate for staining</td>
		</tr>
		<tr>
			<td>FACSAria IIu Cell Sorter</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer</td>
		</tr>
		<tr>
			<td>FCS Express 6</td>
			<td>De Novo Software</td>
			<td></td>
			<td>FACS analysis</td>
		</tr>
		<tr>
			<td>Graphpad Prism</td>
			<td>GraphPad software</td>
			<td></td>
			<td>Data analysis</td>
		</tr>
	</tbody>
</table>

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.

Setup and analysis of a flow cytometry experiment of human peripheral blood cells stained with seven lineage markers (anti-CD3, -CD4, -CD8, -CD14, -CD19, -CD56 and -TCR &#947;&#948; antibodies) using only two fluorochromes are presented.
Representative results are described for anti-CD8 and -CD56 antibody titration. For each antibody (in this example, anti-CD8), data from ten successive 2-fold dilutions were recorded to calculat...

Watch this Scientific Journal Video about Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry at JoVE.com

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

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. ...

The overall goal of this methodology is to expand beyond the optical limit of most flow cytometers by allowing a researcher to record five additional markers to interrogate complex cell populations. Using this technique, it is possible to achieve a deep immunophenotyping when working with instrument with a low number of detectors or sample with limited number of cells. Begin this procedure by carefully transferring drawn blood into a 50-milliliter conical tube.Dilute the blood with an equal amount of PBS without calcium and magnesium. Add 15 milliliters of density gradient medium to the bottom of a new 50-milliliter conical tube. Carefully overlay the diluted blood on top of the density gradient medium, avoiding any mixing between the density gradient medium and diluted blood.Centrifuge at 400 times g for 30 minutes at room temperature with no brake to avoid disruption of the interface. After centrifugation, use a pipette to carefully aspirate and discard the upper layer, paying attention to not remove cells at the interface between the plasma and density gradient medium. Collect as many peripheral blood mononuclear cells, or PBMCs, as possible from the interface without touching the red cell pellet at the bottom of the 50-milliliter conical tube, and transfer to a new tube.Add PBS to bring the final volume to 25 milliliters, and invert several times to mix. Then, wash the PBMCs twice as indicated in the text. After removing the platelets and counting the PBMCs as described in the text, resuspend the cells in PBS, 0.1%sodium azide to a concentration of one times 10 to the seventh cells per milliliter.To prepare the PBMCs for staining, transfer 100 microliters of each sample to a 96-well V-bottom plate. Centrifuge the plate at 350 times g for three minutes at room temperature. Carefully aspirate the supernatant without disturbing the cell pellet.To each well, add 100 microliters of PBS containing a live/dead fixable dye that reacts with free amine on proteins, and resuspend the PBMC mixture carefully. Subsequently, allow the plate to sit for 10 minutes at room temperature for labeling of dead cells. For each sample, prepare 30 microliters of a mix containing all seven antibodies.At this stage, titrated antibodies against different target molecules and in different fluorochromes can be added as well. Centrifuge the 96-well plate at 350 times g for three minutes at room temperature, and carefully aspirate the supernatant without disturbing the cell pellet. Add the antibody cocktail to each well, and resuspend carefully without generating bubbles.Incubate for 30 minutes at room temperature in the dark. After 30 minutes, add 150 microliters of staining buffer to each well, and centrifuge the plate at 350 times g for three minutes at room temperature. Carefully aspirate the supernatant without disturbing the cell pellet, and resuspend the cells in 200 microliters of PBS.The cells are now ready for flow cytometry analysis. Anti-CD3, CD8, CD14, CD19, and TCR gamma delta antibodies are titrated with a maximum staining index curve. Antibody titration is the most critical step in this protocol to properly identify seven immune cell subset using two fluorochromes.To prepare a two-fold antibody dilution, fill 10 wells of a 96-well plate with 40 microliters of staining buffer per well. For the purposes of this video, only the titration of anti-CD8 will be shown. In the first well, increase the final volume to 80 microliters of staining buffer, and add anti-CD8 at a concentration four times the concentration suggested by the manufacturer.Mix well and transfer 40 microliters to the second well. Mix well and repeat this step for all the other wells. Stain 10 samples of PBMCs or whole blood with 30 microliters of the 10 different two-fold dilutions of anti-CD8 following the protocol demonstrated earlier.Acquire data with a flow cytometer, and plot the signal from each dilution. After calculating the stain index for each antibody concentration as detailed in the text, plot the stain index versus the antibody concentration expressed as a fraction of the antibody dilution, and identify the concentration of antibody with the maximum stain index value. To titrate the anti-CD4 and anti-CD56 antibodies, start with the two-fold dilution strategy demonstrated earlier, adding additional concentrations in between to finely identify the range of concentration that will allow separation of CD4-positive T cells and NK cells from the other cell populations.Titrate the anti-CD4 antibody by placing the anti-CD4 signal between the double CD8-positive, CD3-positive signal and the CD3 single positive population. Titrate the anti-CD56 antibody following a strategy similar to the anti-CD4 antibody titration, by placing NK cells between the CD3-negative and the CD3-positive populations. To begin this two-fluorochrome, seven-marker gating strategy, select the lymphocyte gate, and create a dot plot with one of the two fluorochromes used in this protocol on each axis.Gate on CD8-positive T cells identified as CD3 and CD8 double positive cells at the top right corner of the dot plot. Exclude the dim CD8 population which might contain NKT cells. Gate on CD4-positive T cells identified as the population in between CD8-positive T cells and the CD3 single positive populations.Gate on gamma delta T cells identified as high CD3 cells. Subdivide gamma delta T cells to CD8 positive and CD8 negative. Gate on NK cells identified as the population in between CD3-positive and CD3-negative cells.Subdivide the NK cells to CD8 positive and CD8 negative. Gate on B cells identified as the CD3-negative, CD19-positive population on the lower right corner of the dot plot. Select the monocyte gate, and create a dot plot with one of the two fluorochromes used in this protocol on each axis.Gate on the CD3-negative, CD14-positive population. Using this strategy, lymphocytes from a patient with multiple myeloma were gated on the basis of their forward scatter and side scatter and their flow cytometric profile. CD8-positive memory subpopulations and naive T cells were identified by the expression of CD45RA and CCR7.Expression of HLA-DR and CD57 was used to study T cell activation in CD8-positive naive, memory, central memory, effector memory, and effector memory CD45RA-positive cells. In a similar manner, CD4-positive naive and memory T cells were identified. Within the memory population, CCR4 and CCR6 were used to identify T helper subpopulations in CD4-positive T cells.Subpopulations of NK cells were characterized by CD16 and CD57 expression. This strategy was used to investigate the dynamics in immune populations of longitudinal samples from a donor with multiple myeloma receiving a stem cell transplant. Characterization of CD8 and CD4 subpopulations over time showed a sustained CD4-positive and CD8-positive T cell activation and a skewing of T helper to a T-helper-one phenotype.But at day 60, the percentage of B cells dramatically augmented, predicting the patient relapse. Proper cell preparation and antibody titration are critical steps to achieve reliable results using this technique. Antibody targeting other markers can be added to accomplish deeper flow cytometry analysis and to create modular flow cytometric panels targeting several lineages at the same time.Similar approaches aimed at expanding the number of recordable markers could also be developed using different sets of marker or for use in different animal models. Do not forget that sodium azide can cause burns to skin and eyes. Therefore, a lab coat, protective glasses, and gloves should always be worn when handling this reagent.

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Research

JoVE Journal

Immunology and Infection

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