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

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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

1. 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 a....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 cm Graefe forceps (straight and serrated)	Dimeda
11 cm Iris scissors (straight with sharp/sharp heads)	Dimeda
15 mL centrifuge tube	Thermo Fisher Scientific	339650
5 mL syringe	TERUMO
6 cm Petri dish	α-plus
70 µm nylon cell strainer	SPL Lifesciences	93070
Ammonium chloride (NH4Cl)	Sigma	A9434
Anti-mouse CD25	Biolegend	102007	Clone:PC61
Anti-mouse CD4	Biolegend	100453	Clone:GK1.5
Anti-mouse CD44	Biolegend	103029	Clone:IM7
Anti-mouse CD62L	Biolegend	104407	Clone:MEL-14
Anti-mouse CD8	Biolegend	100713	Clone:53-6.7
Anti-mouse TCRβ	Biolegend	109233	Clone:H57-579
BD LSRFortessa	BD Biosciences
Ethylenediaminetetraacetic acid (EDTA)	Bio basic	6381-92-6
FALCON 5ml Polystyrene Round-Bottom Tube (FACS tube)	BD Biosciences	352052
Fetal Bovine Serum (FBS)	Hyclone	ATD161145
Flowjo, LLC	BD Biosciences
Hydroxyethyl piperazineethanesulfonic acid (HEPES)	Sigma	H4034
L-glutamine (200 mM)	Gibco	A2916801
LysoTracker Green DND26	Thermo Fisher Scientific	L7526
MEM Non-essential amino acids (100X)	Gibco	11140050
MitoTracker Green FM	Thermo Fisher Scientific	M7514
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140122
Potassium bicarbonate (KHCO3)	J.T.baker	298-14-6
Propidium iodide solution	Sigma	25535-16-4
RPMI 1640 medium (powder)	Gibco	31800089
Sodium bicarbonate (NaHCO3)	Sigma	S5761
Sodium pyruvate (100 mM)	Gibco	11360070