Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present a protocol to accurately and reliably measure metabolites in rare cell types. Technical improvements, including a modified sheath fluid for cell sorting and the generation of relevant blank samples, enable a comprehensive quantification of metabolites with an input of only 5000 cells per sample.

Abstract

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule intermediates. However, obtaining accurate and reliable measurements of cellular metabolism, particularly in rare cell types like hematopoietic stem cells, has traditionally required pooling cells from multiple animals. A protocol now enables researchers to measure metabolites in rare cell types using only one mouse per sample while generating multiple replicates for more abundant cell types. This reduces the number of animals that are required for a given project. The protocol presented here involves several key differences over traditional metabolomics protocols, such as using 5 g/L NaCl as a sheath fluid, sorting directly into acetonitrile, and utilizing targeted quantification with rigorous use of internal standards, allowing for more accurate and comprehensive measurements of cellular metabolism. Despite the time required for the isolation of single cells, fluorescent staining, and sorting, the protocol can preserve differences among cell types and drug treatments to a large extent.

Introduction

Metabolism is an essential biological process that occurs in all living cells. Metabolic processes involve a vast network of biochemical reactions that are tightly regulated and interconnected, allowing cells to produce energy and synthesize essential biomolecules1. To understand the function of metabolic networks, researchers measure the levels of small molecule intermediates within cells. These intermediates serve as important indicators of metabolic activity and can reveal critical insights into cellular function.

Mass spectrometry (MS) is the most popular choice for the specific detection of metabolites in comple....

Protocol

Breeding and husbandry of all mice used for this protocol were conducted in a conventional animal facility at the Max Planck Institute for Immunobiology and Epigenetics (MPI-IE) according to the regulations of the local authorities (Regierungspräsidium Freiburg). Mice were euthanized with CO2 and cervical dislocation by FELASA B-trained personnel following guidelines and regulations approved by the animal welfare committee of the MPI-IE and the local authorities. No animal experimentation was performed, a.......

Representative Results

FACS sorting enables the isolation of clean populations of different cell types from the same cell suspension (Figure 2 and Figure 3). The specificity of this method relies on the staining of the different cell types with specific surface markers (for example, B cells and T cells from the spleen) or specific combinations of surface markers (for example HSCs and MPPs). Staining of intracellular markers typically requires permeabilization of the cell membrane. Thi.......

Discussion

The most critical steps for successful implementation of targeted metabolomics using this protocol are 1) a robust staining and gating strategy that will yield clean cell populations 2) precise handling of liquid volumes, 3) reproducible timing of all experimental steps, in particular all steps prior to metabolite extraction. Ideally, all samples belonging to one experiment should be processed and measured in one batch to minimize batch effects22. For larger experiments, we suggest collecting cell.......

Acknowledgements

The authors would like to thank the animal facility of the Max Planck Institute of Immunobiology and Epigenetics for providing the animals used in this study.

....

Materials

NameCompanyCatalog NumberComments
13C yeast extractIsotopic SolutionsISO-1
40 µm cell strainerCorning352340
Acetonitrile, LC-MS gradeVWR83640.32
ACK lysis bufferGibco104921Alternatively: Lonza, Cat# BP10-548E
Adenosine diphosphate (ADP)Sigma AldrichA2754
Adenosine monophosphate (AMP)Sigma AldrichA1752
Adenosine triphosphate (ATP)Sigma AldrichA2383
Ammonium Carbonate, HPLC gradeFisher ScientificA/3686/50
Atlantis Premier BEH Z-HILIC column (100 x 2.1 mm, 1.7 µm)Waters186009982
B220-A647Invitrogen103226
B220-PE/Cy7BioLegend103222RRID:AB_313005
CD11b-PE/Cy7BioLegend101216RRID:AB_312799
CD150-BV605BioLegend115927RRID:AB_11204248
CD3-PEInvitrogen12-0031-83
CD48-BV421BioLegend103428RRID:AB_2650894
CD4-PE/Cy7BioLegend100422RRID:AB_2660860
CD8a-PE/Cy7BioLegend100722RRID:AB_312761
cKit-PEBioLegend105808RRID:AB_313217
Dynabeads Untouched Mouse CD4 Cells Kit Invitrogen11415D
FACSAria IIIBD
Gr1-PE/Cy7BioLegend108416RRID:AB_313381
Heat sealing foilNeolabJul-18
IsoleucineSigma Aldrich58880
JetStream ESI SourceAgilentG1958B
LeucineSigma AldrichL8000
Medronic acidSigma AldrichM9508-1G
Methanol, LC-MS gradeCarl RothHN41.2
NaClFluka31434-1KG
PBSSigma AldrichD8537
Sca1-APC/Cy7BioLegend108126RRID:AB_10645327
TER119-PE/Cy7BioLegend116221RRID:AB_2137789
Triple Quadrupole Mass SpectrometerAgilent6495B
Twin.tec PCR plate 96 well LoBind skirtedEppendorf30129512
UHPLC AutosamplerAgilentG7157B
UHPLC Column ThermostatAgilentG7116B
UHPLC PumpAgilentG7120A
UHPLC Sample ThermostatAgilentG4761A

References

  1. Jang, C., Chen, L., Rabinowitz, J. D. Metabolomics and isotope tracing. Cell. 173 (4), 822-837 (2018).
  2. Lu, W., Su, X., Klein, M. S., Lewis, I. A., Fieh, O., Rabinowitz, J. D. Metabolite measurement: Pitfalls to avoid and practices to follow.

Explore More Articles

MetabolomicsRare Primary CellsHematopoietic Stem CellsDietary InterventionsStemnessSingle Cell MethodsLow Input MetabolomicsMass SpectrometryMetabolite DetectionCellular MetabolismLipidomicsTargeted QuantificationInternal Standards

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved