A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Summary

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

Abstract

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.

To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

Introduction

Higher organisms contain complex communities of distinct cell types that collaborate to bring about more complex functions. For example, tumors contain not only cancerous cells, but also fibroblasts, cells that constitute blood vessels, and often immune cell infiltrates¹; blood contains a complex mixture of dozens of immune cell subtypes²; and even cultured cell lines may consist of multiple subpopulations, such as the luminal and basal subtypes of breast cancer cells³. Moreover, distinct cell types that coexist can exhibit metabolic "collaboration". For example, in the brain, astrocytes a....

Protocol

1. Metabolite Extraction

1. Extraction from dish
   1. Culture cells in a 6-well plate in triplicates in stable isotope labeled culture media + dialyzed supplements (serum or other growth supplements) until cells become 75% confluent.
      NOTE: Here culture HeLa cells for 48 h in RPMI containing 40% U-¹³C-Glucose and 70% U-¹³C, ¹⁵N2-Glutamine and 5% dialyzed FBS (Fetal Bovine Serum). Dialyzed FBS is used to get rid of the small molecular weight metabolites which might contaminate the labeled media. Culturing cells in dialyzed supplement prior to the real experiment is recommended to ensure cells are gr....

Results

As an example, here we describe an experiment investigating the metabolism of HeLa cells sorted according to cell cycle phase. To label a wide range of central metabolites on both carbons and nitrogens, we cultured cells for 48 h using U-¹³C-glucose and U-¹³C, ¹⁵N-glutamine as tracers. To obtain rich MIDs for the validation experiment, we here chose a mixture of 40% U-¹³C-Glucose and 70% U-¹³C,¹⁵N2-Glutamine, as intermediat.......

Discussion

Our method is based on the principle that MIDs in cellular metabolites reflect the "history" of metabolic activities of a cell. This makes it possible to investigate metabolic activities in subpopulation of cells, as they occurred in the complex community of cells, prior to the cell sorting procedure. In contrast, peak areas of metabolites differ markedly between extracts of sorted cells and direct extraction from the culture dish¹¹. In part this is because the different chemical compositi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HBSS	Sigma	H6648
INFLUX (inFlux v7 Sorter)	BD Biosciences
U-13C-Glucose	Cambridge isotopes	40762-22-9 / GLC-018
U-13C,15N2-Glutamine	Cambridge isotopes	CNLM-1275-H-0.1
Methanol (JT Baker), HPLC grade	VWR	BAKR8402.2500
Ultrafree - MC - VV centrifugal Filters. Durapore PVDF 0.1 µm	Millipore	UFC30VV00
Ultimate 3,000 UHPLC	Thermo Fisher scientific
Q-Exactive Orbitrap Mass spectrometer	Thermo Fisher scientific
Merk-Sequant ZIC HILIC column (150 mm x 4.6 mm, 5 µm)	Merck KGaA	1.50444.0001
Merk-Sequant ZIC HILIC guard column (20 mm x 2.1 mm)	Merck KGaA
Acetonitrile Optima LC-MS, amber glass	Fisher Scientific	A955-212
Milli-Q water	Millipore		Produced with a Milli-Q Gradient system
Myrsyra 99.5% Optima (Formic acid)	Fisher Scientific	11423423
X100 Screw Vial 1.5 ml, 8-425 32x11.6 mm, amber, 100 units	Thermo Fisher scientific	10560053
X100 Lock Skruv Vitt PTFE Packing 8-425 (Screw caps)	Thermo Fisher scientific	12458636
ProteoMass LTQ/FT-Hybrid ESI Pos. Mode Cal Mix	Sigma-Aldrich	MSCAL5	Calibration kit
SNAKESKIN 10K MWCO	Thermo Fisher scientific	88245
Mathematica v.10	Wolfram Research