The development of this protocol was supported by grants from the Taiwan Ministry of Science and Technology (MOST) NSC103-2320-B-010-002-MY2 and MOST104-2628-B-010-002-MY4 to C.L. Hsu. C.W. Wei is a recipient of Excellent Thesis Award of Institute of Microbiology and Immunology, National Yang-Ming University.

The mouse tissue harvest procedure described here has been approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang-Ming University.
1. Preparation of Lymphocyte Suspension from Lymphoid Organs
<ol>
	<li>Euthanize the mouse by an approved method such as CO2 inhalation in a transparent acrylic chamber followed by cervical dislocation to ensure death. 
	NOTE: Keep the flow rate of CO2 for a 20% displacement per minute of the chamber, and no higher than 5 psi (pound per square inch). It takes 2-3 min for a mouse to lose consciousness. Maintain the CO2 flow at least for 1 min after the animal has stopped breathing (5 - 7 min overall).</li>
	<li>Place the mouse on its back on a clean board (e.g. a polystyrene plastic board) and fix the limbs with tape. Rinse with 70% ethanol.</li>
	<li>To harvest spleen, cut and open the abdominal cavity with 11-cm dissecting scissors (the spleen is below the stomach and above the left kidney). Remove the spleen with forceps and clean from connective tissues (use scissors if necessary).</li>
	<li>To harvest the thymus, cut the diaphragm and the rib cage from both sides with dissecting scissors. Use forceps to lift up the sternum to reveal the entire thoracic cavity. Separate the thymus carefully from surrounding tissues with scissors, and remove any residual connective tissue on a paper towel wetted with PBS.</li>
	<li>Mechanically dissociate the tissues with a syringe plunger in a 6-cm dish containing 5 mL ice-cold FACS buffer (PBS supplemented with 2% fetal bovine serum (FBS) and 1 mM EDTA). Transfer the single-cell suspension to a 15-mL centrifuge tube; wash the 6-cm dish with additional 2 mL FACS buffer and combine the wash with cell suspension as well.</li>
	<li>Centrifuge the cell suspension (300 x g, for 5 min at 4 &#176;C) and discard the supernatant. Re-suspend the pellet with 2 mL of Ammonium-Chloride-Potassium (ACK) lysing buffer (155 mM NH4Cl, 10 mM KHCO3 and 0.1 mM Na2EDTA) for 1 min at room temperature (RT) to lyse the red blood cells. Neutralize the ACK buffer by adding 10 mL FACS buffer to the cell suspension.</li>
	<li>Centrifuge the cell suspension (300 x g, for 5 min at 4 &#176;C) and discard the supernatant. Re-suspend cells in 5 mL of complete RPMI medium (RPMI 1640 medium, supplemented with 44 mM NaHCO3, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 U/mL penicillin, 100 mg/mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% MEM non-essential amino acids and 10% FBS).</li>
	<li>Filter cell suspension through a cell strainer (pore size 70 &#181;m) to remove cell clumps. Count cells with a hemocytometer or automated cell counter.</li>
</ol>
2. Quantification of Mitochondria and Lysosomes
NOTE: Perform the organelle-specific dye staining first because the optimal staining condition for these dyes is incubation at 37 &#176;C. The surface marker staining can be significantly reduced because of antibody capping and internalization at that temperature.
<ol>
	<li>Add 1 x 106 cells to round-bottom FACS tubes, centrifuge cells (300 x g, for 5 min at 4 &#176;C) and discard supernatant.</li>
	<li>Dilute the probe stock solutions to final working concentration (100 nM MitoTracker Green or LysoTracker Green; henceforth mitochondria- or lysosome-specific dye, respectively)&#160;in pre-warmed 37 &#176;C serum-free culture medium. This is the working solution for lysosome- or mitochondria-specific dye. 
	NOTE: Test multiple concentrations including the range of working concentrations suggested by manufacturer (20 - 200 nM for the mitochondria-specific dye and 50 - 75 nM for the lysosome-specific dye used here) in order to obtain the best staining efficiency. Choose a cell population that is known to have good expression of staining target (e.g. lysosomes) as positive control (e.g. human CD14+ monocytes). Choose the working concentration based on good separation between negative and positive staining, as well as good cell viability.</li>
	<li>Resuspend the cells in 100 &#181;L of lysosome- or mitochondria-specific dye working solution and incubate in 5% CO2 incubator at 37 &#176;C for 15 min or 30 min, respectively.</li>
	<li>Add 1 mL of ice-cold FACS buffer to stop the reaction. Pellet cells by centrifugation (300 x g, for 5 min at 4 &#176;C) and discard supernatant. Cells are now ready for surface marker staining.</li>
</ol>
3. Surface Marker Staining
<ol>
	<li>Block Fc receptors with 100 &#181;L of pre-titrated 2.4G2 hybridoma supernatant on ice for 10 min. Wash cells with 1 mL of FACS buffer and centrifuge (300 x g, for 5 min at 4 &#176;C). Discard supernatant.</li>
	<li>Incubate the cells with 50 &#181;L pre-titrated fluorescence-conjugated antibody combination in FACS buffer on ice for 20 min. Avoid light. 
	NOTE: Prepare single color-stained cells that include all the fluorescent antibodies present in the antibody combination and cells treated only with organelle-specific dye for setting voltages of each fluorescence detector and compensation adjustment on flow cytometer. Likewise, prepare the Fluorescence Minus One (FMO) as background controls by using cells stained with all the antibodies, but without staining with organelle-specific dyes.</li>
	<li>Wash the cells by adding 1 mL of FACS buffer and pellet by centrifugation (300 x g, for 5 min at 4 &#176;C). Discard supernatant.</li>
	<li>Resuspend the cells in 300 &#181;L of FACS buffer containing 1 &#181;g/mL propidium iodide (PI) and immediately analyze the samples on flow cytometer.</li>
</ol>
4. Sample Collection on Flow Cytometer
<ol>
	<li>Turn on the computer, flow cytometer, FACS acquisition software and any other accessory devices depending on the machine platform. Run PBS for around 1 min to ensure the flow line is filled with PBS and sheath buffer. Make sure the flow stream runs smoothly.</li>
	<li>Open new experiment and select cytometer parameters (fluorescent detectors) according to manufacturer&#39;s instruction. Filter cells through 70-&#181;m cell strainer and vortex prior to the acquisition of each sample to avoid clumps and doublet formation.</li>
	<li>Make dot plots of forward scatter (FSC) and side scatter (SSC), which represent cell size and granularity, respectively. Optimize the voltages of FSC and SSC to make sure the cells of interest appear in the plots. Make dot plots of FSC-A vs. FSC-W (or FSC-H) and draw a gate for the single cells.</li>
	<li>Make dot plots of each fluorescent parameter. Use unstained cells to determine background signal and use single color-stained controls to adjust voltages of each fluorescent parameter, so that positive and negative cell populations are clearly distinguishable and within the dynamic range of acquisition.</li>
	<li>After all the voltages are set, use auto-compensation if available, or manually adjust the compensation with unstained and single color-stained controls to correct the spectral spillover. Apply the compensation to samples.</li>
	<li>Make a dot plot of each parameter and draw gates according to your experimental set up. For example, FSC-A vs. FSC-H (or FSC-W) to exclude doublets, followed by FSC-A vs. SSC-A for lymphocyte gating; SSC-A vs. PI (live cells) and CD4 vs. CD8 (Figure 1).</li>
	<li>Collect enough events (total cell number) for meaningful comparison between populations. Typically, at least 1,000 events in the population of interest need to be obtained20.</li>
	<li>After all the samples are acquired, export flow data to be analyzed by flow cytometry analysis software. Save the experiment as template to preserve the cytometer setting and parameters for applying to similar experiments in the future.</li>
</ol>
5. Data Analysis
NOTE: There are many tools to analyze flow cytometric data, both commercial and free software. The acquisition software of the flow cytometers can also work for data analysis. Here we used FlowJo as an example.
<ol>
	<li>Start the software and drag the FCS files into the workspace. Click on a sample to open a graphic window. Select parameters to be presented on X and Y axis by clicking on the X and Y axes. Use the toolbar to draw gates according to differentially expressed markers or defined phenotypes of cell populations.</li>
	<li>Add gates of each parameter as shown in Figure 1. Drag gates to all samples in the workspace, so that they get identical gating.</li>
	<li>Drag the plots to layout editor to create graphical reports.</li>
	<li>Display the mean fluorescence intensity (MFI) for each population of interest by clicking the statistics button (&#931;) in a graphic window and choosing &#34;Mean&#34; and the appropriate parameter, for example, the channel used to detect organelle-specific dye. Then click &#34;Add&#34;.</li>
	<li>Click the table editor icon and drag MFI to table editor to create statistical reports. Save as Excel, Text or CSV file to calculate delta MFI (&#916;MFI) as MFI (organelle-specific dyes) - MFI (FMO). 
	NOTE: Do not calculate MFI between samples acquired with different cytometer settings (voltages, fluorescent channels or compensation). Comparisons of values derived from experiments with different settings are meaningless and biased.</li>
	<li>Export all the values from the workspace and plots from layout editor for making tables and figures.</li>
</ol>

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The authors declare no conflicts of interests.

This protocol combines organelle-specific dyes and surface marker staining to quantify the amount of mitochondria or lysosomes in different T cell populations. This method was developed to overcome the limitation of cell number and homogeneity requirements for traditional methods, such as electron microscopy and immunoblot analysis. It is especially useful in analyzing rare cell populations and simultaneously examining multiple cell types at the same time.
The duration of the procedure and the...

The activation and proliferation of T cells are critical steps for mounting successful immune responses. Recent advances suggest that the cellular metabolism is tightly associated with both the development and functions of T cells. For example, naive T cells rely largely on oxidative phosphorylation (OXPHOS) to meet the energy demand during the recirculation among secondary lymphoid organs. Upon activation, naive T cells undergo drastic metabolic reprogramming, including the induction of aerobic glycolysis to increase ATP production and to fulfill the tremendous metabolic demands during cell differentiation and proliferation. The cells that fail to follow through the metabolic needs die by apoptosis1,2. During the metabolic reprogramming, mitochondria play important roles since they are the organelles largely responsible for the production of ATP to supply energy for the cell, and the cellular mitochondria content fluctuates during metabolic switches throughout T cell development and activation3. However, the accumulation of superfluous or damaged mitochondria can produce excess ROS that damage lipids, proteins, and DNA, and can eventually lead to cell death4
The excessive or damaged mitochondria resulting from metabolic alterations are removed by a specialized form of autophagy5, known as mitophagy. Mitochondria are wrapped by autophagosomes and then fused with lysosomes for degradation. These close communications between mitochondria and lysosomes have generated great interest6,7. For example, oxidative stress stimulates mitochondria to form mitochondria-derived vesicles (MDVs) that are targeted to lysosomes for degradation in a phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and parkin (an E3 ubiquitin ligase) dependent manner8. It was also found that mitophagy is essential for beige-to-white adipocyte transition9,10. More importantly, lysosomes are not merely a degradation compartment, but also a regulator of cellular signaling. Excessive substrate accumulation due to enzyme deficiencies results in lysosomal dysfunction by disrupting lysosomal membrane permeability and affects Ca2+ homeostasis11. The T cell functional defects in lysosomal acid lipase (LAL)12 or lysosomal metabolite transporter knockout mouse model13 further showed the importance of lysosomes in maintaining T cell homeostasis. Both mitochondria and lysosomes are inseparable parts of cellular metabolic regulation. Therefore, measurement of cellular mitochondrial content has been a crucial indicator to evaluate the T cell metabolic and functional status.
Assays commonly used to quantify cellular mitochondrial or lysosomal contents include immunoblot, electron microscopy, immunofluorescent (IF) staining, and PCR analysis of mitochondrial DNA copy numbers14,15,16. While immunoblot can quantitatively compare protein levels across different samples and electron or IF microscopy can visualize the morphological hallmarks of these organelles17, these assays bear certain technical drawbacks. For example, it is time consuming to acquire sufficient number of cell images with high magnification and resolution or to compare the expression levels of a protein across dozens of samples, making these assays considered to be low throughput methods. Moreover, these assays can only be applied to homogenous cell population, such as cell lines, but not to tissue samples that are composed of mixed populations.
It is also difficult to apply these assays to rare populations, for which the requirement of minimal cell numbers from 106 to 108 is impossible to meet. Finally, cells are usually lysed or fixed during the process, making them incompatible with other methods to extract further information. Compared with the traditional methods, fluorescent-based flow cytometry has a relatively high throughput - the information of all the cells within a sample composed of mixed cell populations can be analyzed and collected simultaneously. In addition, one can detect more than 10 parameters on the same cell and sort the cells based on desired phenotypes for further assays. Reactive fluorescent probes have been used to label lysosomes and mitochondria in live cells and can be detected by flow cytometry18,19. These organelle-specific probes are cell permeable and have physico-chemical characteristics that allow them to be concentrated in specific subcellular locations or organelles. Conveniently, these probes are available in various fluorescent formats, thus enabling their application for multicolor analysis.
This protocol describes in detail how to combine surface marker staining with lysosome- or mitochondria-specific dyes to label lysosomes or mitochondria in live cells. This is especially useful for samples generated from primary tissues and organs, which are often composed of heterogeneous cell populations. Researchers can identify cell populations of interest by their surface marker expression and specifically measure the lysosomal or mitochondrial contents through organelle-specific dyes in these cells. Here, we demonstrate the detailed procedure of flow cytometric analysis that evaluates the lysosomal or mitochondrial mass in the major splenic T cell subpopulations.

<table border="0" cellpadding="0" cellspacing="0" style="width:763px;" width="762">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">10 cm Graefe forceps (straight and serrated)</td>
			<td style="width:179px;">Dimeda</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">11 cm Iris scissors (straight with sharp/sharp heads)</td>
			<td>Dimeda</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">15 mL centrifuge tube</td>
			<td style="width:179px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">339650</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">5 mL syringe</td>
			<td style="width:179px;">TERUMO&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">6 cm Petri dish</td>
			<td style="width:179px;">&#945;-plus</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">70 &#181;m nylon cell strainer</td>
			<td style="width:179px;">SPL Lifesciences</td>
			<td style="width:119px;">93070</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Ammonium chloride (NH4Cl) &#160;&#160;</td>
			<td>Sigma</td>
			<td style="width:119px;">A9434</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-mouse CD25</td>
			<td style="width:179px;">Biolegend</td>
			<td style="width:119px;">102007</td>
			<td style="width:167px;">Clone:PC61</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-mouse CD4</td>
			<td style="width:179px;">Biolegend</td>
			<td style="width:119px;">100453</td>
			<td style="width:167px;">Clone:GK1.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-mouse CD44</td>
			<td style="width:179px;">Biolegend</td>
			<td style="width:119px;">103029</td>
			<td style="width:167px;">Clone:IM7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-mouse CD62L</td>
			<td style="width:179px;">Biolegend</td>
			<td style="width:119px;">104407</td>
			<td style="width:167px;">Clone:MEL-14</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-mouse CD8</td>
			<td style="width:179px;">Biolegend</td>
			<td style="width:119px;">100713</td>
			<td style="width:167px;">Clone:53-6.7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-mouse TCR&#946;</td>
			<td style="width:179px;">Biolegend</td>
			<td style="width:119px;">109233</td>
			<td style="width:167px;">Clone:H57-579</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">BD LSRFortessa</td>
			<td style="width:179px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:179px;">Bio basic</td>
			<td style="width:119px;">6381-92-6</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">FALCON 5ml Polystyrene Round-Bottom Tube (FACS tube)</td>
			<td style="width:179px;">BD Biosciences</td>
			<td style="width:119px;">352052</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">FcRgamma II (CD32) Hybridoma (2.4G2)</td>
			<td style="width:179px;">ATCC</td>
			<td style="width:119px;">HB-197&#12288;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:179px;">Hyclone</td>
			<td>ATD161145</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Flowjo, LLC</td>
			<td style="width:179px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydroxyethyl piperazineethanesulfonic acid (HEPES)</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine (200 mM)</td>
			<td style="width:179px;">Gibco</td>
			<td>A2916801</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">LysoTracker Green DND26</td>
			<td style="width:179px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">L7526</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">MEM Non-essential amino acids (100X)</td>
			<td style="width:179px;">Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">MitoTracker Green FM</td>
			<td style="width:179px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">M7514</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:179px;">Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium bicarbonate (KHCO3)</td>
			<td style="width:179px;">J.T.baker</td>
			<td style="width:119px;">298-14-6</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Propidium iodide solution</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:119px;">25535-16-4</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">RPMI 1640 medium (powder)</td>
			<td style="width:179px;">Gibco</td>
			<td>31800089</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Sodium pyruvate (100 mM)</td>
			<td style="width:179px;">Gibco</td>
			<td>11360070</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

multicolor flow cytometry-based quantification of mitochondria and lysosomes in t cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

Identification of major T cell subsets in spleen and thymus
In brief, single cell suspensions from spleen and thymus were lysed of red blood cells, incubated with 2.4G2 supernatant, followed by organelle-specific dye and surface marker staining with fluorescence-conjugated antibodies (Table 1). The developmental progression of thymocytes can be described by using antibodies to the co-receptors C...

Watch this Scientific Journal Video about Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells at JoVE.com

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

multicolor-flow-cytometry-based-quantification-mitochondria-lysosomes

Research

JoVE Journal

Immunology and Infection

12.9K Views.  National Yang-Ming University. This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

Video: Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...