Name: multicolor flow cytometry-based quantification of mitochondria and lysosomes in t cells
Uploaded: 2019-01-09T14:30:00
Description: Watch this Scientific Journal Video about Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells at JoVE.com

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 n...

<ol>
	<li>Buck, M. D., O'Sullivan, D., Pearce, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+metabolism+drives+immunity.">T cell metabolism drives immunity.</a> Journal of Experimental Medicine. 212 (9), 1345-1360 (2015).</li><li>Marelli-Berg, F. M., Fu, H., Mauro, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+metabolic+reprogramming+in+proliferating+cells:+implications+for+T-cell-mediated+immunity.">Molecular mechanisms of metabolic reprogramming in proliferating cells: implications for T-cell-mediated immunity.</a> Immunology. 136 (4), 363-369 (2012).</li><li>Pua, H. H., Guo, J., Komatsu, M., He, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+is+essential+for+mitochondrial+clearance+in+mature+T+lymphocytes.">Autophagy is essential for mitochondrial clearance in mature T lymphocytes.</a> The Journal of Immunology. 182 (7), 4046-4055 (2009).</li><li>Chao, T., Wang, H., Ho, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Control+and+Guidance+of+Cellular+Activities+of+T+Cells.">Mitochondrial Control and Guidance of Cellular Activities of T Cells.</a> Frontiers in Immunology. 8, 473 (2017).</li><li>Youle, R. J., Narendra, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+mitophagy.">Mechanisms of mitophagy.</a> Nature Reviews Molecular Cell Biology. 12 (1), 9-14 (2011).</li><li>Diogo, C. V., Yambire, K. F., Fern&#225;ndez Mosquera, L., Branco, F. T., Raimundo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+adventures+at+the+organelle+society.">Mitochondrial adventures at the organelle society.</a> Biochemical and Biophysical Research Communications. 500 (1), 87-93 (2018).</li><li>Todkar, K., Ilamathi, H. S., Germain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+Lysosomes:+Discovering+Bonds.">Mitochondria and Lysosomes: Discovering Bonds.</a> Frontiers in Cell and Developmental Biology. 5, 106 (2017).</li><li>McLelland, G. L., Soubannier, V., Chen, C. X., McBride, H. M., Fon, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkin+and+PINK1+function+in+a+vesicular+trafficking+pathway+regulating+mitochondrial+quality+control.">Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control.</a> The EMBO Journal. 33 (4), 282 (2014).</li><li>Wrighton, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism:+Mitophagy+turns+beige+adipocytes+white.">Metabolism: Mitophagy turns beige adipocytes white.</a> Nature Reviews Molecular Cell Biology. 17 (10), 607 (2016).</li><li>Altshuler-Keylin, 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=Beige+Adipocyte+Maintenance+Is+Regulated+by+Autophagy-Induced+Mitochondrial+Clearance.">Beige Adipocyte Maintenance Is Regulated by Autophagy-Induced Mitochondrial Clearance.</a> Cell Metabolism. 24 (3), 402-419 (2016).</li><li>Settembre, C., Fraldi, A., Medina, D. L., Ballabio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signals+from+the+lysosome:+a+control+centre+for+cellular+clearance+and+energy+metabolism.">Signals from the lysosome: a control centre for cellular clearance and energy metabolism.</a> Nature Reviews Molecular Cell Biology. 14 (5), 283-296 (2013).</li><li>Qu, P., Du, H., Wilkes, D. S., Yan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+roles+of+lysosomal+acid+lipase+in+T+cell+development+and+function.">Critical roles of lysosomal acid lipase in T cell development and function.</a> The American Journal of Pathology. 174 (3), 944-956 (2009).</li><li>Wei, C. 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=Equilibrative+Nucleoside+Transporter+3+Regulates+T+Cell+Homeostasis+by+Coordinating+Lysosomal+Function+with+Nucleoside+Availability.">Equilibrative Nucleoside Transporter 3 Regulates T Cell Homeostasis by Coordinating Lysosomal Function with Nucleoside Availability.</a> Cell Reports. 23 (8), 2330-2341 (2018).</li><li>Baixauli, 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=Mitochondrial+Respiration+Controls+Lysosomal+Function+during+Inflammatory+T+Cell+Responses.">Mitochondrial Respiration Controls Lysosomal Function during Inflammatory T Cell Responses.</a> Cell Metabolism. 22 (3), 485-498 (2015).</li><li>Piantadosi, C. A., Suliman, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+transcription+factor+A+induction+by+redox+activation+of+nuclear+respiratory+factor+1.">Mitochondrial transcription factor A induction by redox activation of nuclear respiratory factor 1.</a> The Journal of Biological Chemistry. 281 (1), 324-333 (2006).</li><li>Zhu, 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=Constitutive+association+of+the+proapoptotic+protein+Bim+with+Bcl-2-related+proteins+on+mitochondria+in+T+cells.">Constitutive association of the proapoptotic protein Bim with Bcl-2-related proteins on mitochondria in T cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (20), 7681-7686 (2004).</li><li>Ding, W. X., Yin, X. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy:+mechanisms,+pathophysiological+roles,+and+analysis.">Mitophagy: mechanisms, pathophysiological roles, and analysis.</a> Biological Chemistry. 393 (7), 547-564 (2012).</li><li>van der Windt, G. J. 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=CD8+memory+T+cells+have+a+bioenergetic+advantage+that+underlies+their+rapid+recall+ability.">CD8 memory T cells have a bioenergetic advantage that underlies their rapid recall ability.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (35), 14336 (2013).</li><li>Chikte, S., Panchal, N., Warnes, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Lyso+dyes:+A+flow+cytometric+study+of+autophagy.">Use of Lyso dyes: A flow cytometric study of autophagy.</a> Cytometry Part A. 85 (2), 169-178 (2013).</li><li>Allan, A. L., Keeney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cell+analysis:+technical+and+statistical+considerations+for+application+to+the+clinic.">Circulating tumor cell analysis: technical and statistical considerations for application to the clinic.</a> Journal of Oncology. 2010, 426218 (2010).</li><li>Curtsinger, J. M., Lins, D. C., Johnson, C. M., Mescher, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+3+tolerant+CD8+T+cells+degranulate+in+response+to+antigen+but+lack+granzyme+B+to+mediate+cytolysis.">Signal 3 tolerant CD8 T cells degranulate in response to antigen but lack granzyme B to mediate cytolysis.</a> The Journal of Immunology. 175 (7), 4392-4399 (2005).</li><li>Wolint, P., Betts, M. R., Koup, R. A., Oxenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+cytotoxicity+but+not+degranulation+distinguishes+effector+and+memory+subsets+of+CD8++T+cells.">Immediate cytotoxicity but not degranulation distinguishes effector and memory subsets of CD8+ T cells.</a> Journal of Experimental Medicine. 199 (7), 925-936 (2004).</li><li>Van den Bossche, 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=Mitochondrial+Dysfunction+Prevents+Repolarization+of+Inflammatory+Macrophages.">Mitochondrial Dysfunction Prevents Repolarization of Inflammatory Macrophages.</a> Cell Reports. 17 (3), 684-696 (2016).</li><li>Pearce, E. 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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.

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			<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>
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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 ...

Research

JoVE Journal

Immunology and Infection

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