Method to Study Metabolism in Lymphoid Cells using Chemistry to Measure Puromycin Incorporation by Flow Cytometry

Summary

The protocol shows a flow cytometry-based method using chemistry to study energy metabolism in cells.

Abstract

Upon antigen stimulation, naïve T cells undergo rapid proliferation and expansion to effector T cells. Metabolism plays an important role in the generation of biomass needed for these rapidly proliferating cells and for the generation of molecules required for effector T cell differentiation and function, which influence the outcome of the adaptive immune response in infection or cancers. Naïve T cells reprogram their metabolism upon antigenic stimulation to increase the generation of ATP, which is required to support their growth, biosynthesis, and effector functions. ATP can be generated in a cell either by the mitochondrial-oxidative phosphorylation (OXPHOS) pathway or by the glycolytic pathway. Because most of the ATP generated in a dividing, growing cell is used up for the synthesis of proteins, protein synthesis has been used as a surrogate for ATP levels. Protein synthesis can be measured by the incorporation of puromycin, which mimics the 3′ adenosine of a tRNA charged with a modified tyrosine and leads to spontaneous termination of protein translation. Metabolic inhibitors like 2-deoxyglucose (2DG), which blocks the glycolytic pathway, and Oligomycin (O), which blocks complex 5 of the electron transport chain (ETC), can be used to study the dependencies of cellular ATP generation on these two pathways in conjunction with evaluation of protein synthesis in a method called SCENITH. We describe here a variation of this method that detects puromycin incorporation by flow cytometry using chemistry. This method of studying metabolism is relatively easy and can be used for evaluating rare cell populations, as well as patient samples, by flow cytometry.

Introduction

In recent years, there has been a growing appreciation of the important role of metabolism in the regulation of T cells. Cellular metabolism regulates many critical functions in lymphocytes, including clonal expansion of pathogen-specific T cells and their differentiation into effector cell subsets, which synthesize a variety of effector molecules, including inflammatory cytokines and cytolytic granzymes required to kill pathogens¹. The study of T cell metabolism is therefore important both for evaluating normal T cell function and for the development of potential therapeutics.

Naïve T cells are metabolically qu....

Protocol

Animal husbandry and experiments were performed in accordance with approved protocol G98-3 by the National Human Genome Research Institute's Animal Use and Care Committee, NIH, and approved protocol LISB 22E by the National Institute of Allergy and Infectious Diseases Animal Care and Use Committee, NIH. Male C57Bl/6J mice aged 6-12 weeks, bred and housed in specific pathogen-free (SPF) conditions, were used for the present study. Similar results were obtained with female mice. Human whole blood was obtained from the NIH Blood Bank under NIH Clinical Center IRB-approved protocol 99-CC-0168 Collection and Distribution of Blood Components from Healthy Donors for In V....

Results

Naïve CD4⁺ T cells were isolated from the spleen and lymph nodes of mice and differentiated to Th0 or non-polarized (NP) lineages for 72 h. Cells were harvested after 0 h, 48 h, and 72 h of culture, and OPP incorporation was measured by looking at the MFI of the OPP. Naïve CD4⁺ T cells were less synthetic than the activated T cells, as seen by the MFI of OPP (Figure 3). Naïve cells were partially dependent on glycolysis but more so on the OXPHOS pathway as evaluated by .......

Discussion

This paper provides a method to study metabolism in lymphoid cells by flow cytometry, based on SCENITH technology, but using chemical labeling of incorporated puromycin. Although we have focused on lymphocytes, this protocol can be adjusted to study metabolism in any cell type. This method is also advantageous to study metabolism in rare populations of cells and patient samples, where other techniques, such as Seahorse, require larger numbers of cells.

A critical step in the protocol is to mak.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Mercaptoethanol	Thermo Fisher	21985-023
Alexa Fluor 488 anti-Puromycin Antibody	Biolegend	381506	Dilution used: 1/100
Alexa Fluor 647 anti-human CD4 Antibody	Biolegend	357422	Dilution used: 1/50
BD Horizon BUV737 Rat Anti-Mouse CD4	BD Biosciences	612761	Dilution used: 1/200
Click-iT Plus OPP Alexa Fluor 488 Protein Synthesis Assay Kit	Thermo Fisher	C10456
HEPES (1 M)	Thermo Fisher	15630080
Human Serum	Millipore Sigma	H4522
IMDM, GlutaMAX Supplement, 500 ml	Thermo Fisher	31980030
InVivoMAb anti-mouse CD28, Clone: 37.51	BioXcell	BE0015-1
InVivoMAb anti-mouse CD3ε, Clone: 145-2C11	BioXcell	BE0001-1
InVivoMAb anti-mouse IFNγ, Clone: XMG1.2	BioXcell	BE0055
InVivoMAb anti-mouse IL-12 p40, Clone: C17.8	BioXcell	BE0051
InVivoMAb anti-mouse IL-4, Clone: 11B11	BioXcell	BE0045
L-Glutamine (200 mM)	Thermo Fisher	25030081
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation	Thermo Fisher	L34966	Dilution used: 1/600
LSM lymphocyte separation medium	MP Biomedicals	50494X
MEM Non-Essential Amino Acids Solution (100X)	Thermo Fisher	11140050
Mitomycin C from Streptomyces caespitosus	Sigma	M0503-10X2MG
Naive CD4 Isolation Kit, mouse	Miltenyi Biotec	130-104-453
Paraformaldehyde 16% Aqueous Solution EM Grade	Electron Microscopy Sciences	15710
PBS (1X), pH 7.4, 500 mL	Quality Biologicals	114-058-101
Penicillin-Streptomycin (10,000 U/mL). 100mL	Thermo Fisher	15140122
Phytohemagglutinin-L (PHA-L)	Millipore Sigma	11249738001
Premium Grade Fetal Bovine Serum (FBS)	Avantor	97068-085
RPMI 1640 Medium, no glutamine	Thermo Fisher	21870076
Saponin	Thermo Fisher	A18820.22
Sodium Pyruvate (100 mM)	Thermo Fisher	11360070