Name: a method for measuring metabolism in sorted subpopulations of complex cell communities using stable isotope tracing
Uploaded: 2017-02-04T14:00:00
Description: Watch this Scientific Journal Video about A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing at JoVE.com

The authors would like to thank Dr. Anas Kamleh for valuable help with optimizing mass spectrometry methods, and Annika von Vollenhoven for assistance with cell sorting. This research was supported by the Swedish Foundation for Strategic Research (grant no. FFL12-0220) and the Strategic Programme in Cancer Research (IR, RN); the Swedish Heart-Lung Foundation (CEW, HG); and Mary Kay Foundation (JW, MJ).

1. Metabolite Extraction
<ol>
	<li>Extraction from dish
	<ol>
		<li>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 metaboli...

<ol>
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Our method is based on the principle that MIDs in cellular metabolites reflect the &#34;history&#34; 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...

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 &#34;collaboration&#34;. For example, in the brain, astrocytes are thought to convert glucose to lactate, which is then &#34;fed&#34; to neurons that oxidize this substrate4; T lymphocytes are in some contexts dependent on adjacent dendritic cells as a source of cysteine5; and cancer cells may collaborate with associated fibroblasts in tumors6. To understand the metabolic behavior of such systems, it is essential to separate and measure the metabolic activities of the various cell types present.
By far the most widely used method for separating cell types is fluorescence-activated cell sorting. This method is broadly applicable, provided that the cell type or state of interest can be &#34;labeled&#34; using fluorescent antibodies, expression of engineered fluorescent proteins, or other dyes. One option is to initially separate cells types through a cell sorter, re-culture the individual cell types obtained, and then perform metabolism studies of these cultures7. However, this is only feasible if the cell type or phenotype is stable in culture conditions, and cannot capture transient behavior such as cell cycle states, nor the metabolic cooperation in co-cultures. For such cases, metabolism must be measured directly on sorted cells. This is challenging since the cell sorting procedure subjects cells to stresses that may distort their metabolism8, and we are aware of only a few studies taking this approach9,10. In particular, we have found that major metabolites such as amino acids may leak from cells kept in cell sorting buffer, so that measurements of absolute metabolite abundance are no longer reliable11 (although relative comparison between sorted fractions may still be valuable).
To circumvent these issues, we label cells with stable isotopes prior to sorting, and focus on the MIDs in cellular metabolites, rather than metabolite abundances. Since MIDs are formed over longer time scales, they should be less affected by short-term exposure to sorting conditions. We quantify MIDs using full-scan high-resolution mass spectrometry, which is sensitive enough to provide data on hundreds of metabolites starting from around 500,000 sorted cells, requiring about 30-60 min of cell sorting time. A comparison between a &#34;mock sorted&#34; control (cells passed through the cell sorter instrument without gating any specific population) and metabolite extraction directly from the culture dish is made to ensure that the observed MIDs are representative of those in the original culture. Depending on the choice of stable isotope tracers, various metabolic pathways can be studied with this method.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">HBSS</td>
			<td style="width:165px;">Sigma</td>
			<td style="width:139px;">H6648</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">INFLUX (inFlux v7 Sorter)</td>
			<td style="width:165px;">BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">U-13C-Glucose</td>
			<td>Cambridge isotopes</td>
			<td>40762-22-9 / GLC-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">U-13C,15N2-Glutamine</td>
			<td>Cambridge isotopes</td>
			<td>CNLM-1275-H-0.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol (JT Baker), HPLC grade</td>
			<td style="width:165px;">VWR</td>
			<td style="width:139px;">BAKR8402.2500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrafree - MC - VV centrifugal Filters. Durapore PVDF 0.1 &#181;m</td>
			<td style="width:165px;">Millipore</td>
			<td style="width:139px;">UFC30VV00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultimate 3,000 UHPLC</td>
			<td style="width:165px;">Thermo Fisher scientific</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Q-Exactive Orbitrap Mass spectrometer</td>
			<td style="width:165px;">Thermo Fisher scientific</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Merk-Sequant ZIC HILIC column (150 mm x 4.6 mm, 5 &#181;m)</td>
			<td style="width:165px;">Merck KGaA</td>
			<td style="width:139px;">1.50444.0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Merk-Sequant ZIC HILIC guard column (20x2.1 mm)</td>
			<td style="width:165px;">Merck KGaA</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetonitrile Optima LC-MS, amber glass</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:139px;">A955-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Milli-Q water</td>
			<td style="width:165px;">Millipore</td>
			<td style="width:139px;"></td>
			<td>Produced with a Milli-Q Gradient system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MYRSYRA 99.5% OPTIMA (Formic acid)</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:139px;">11423423</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X100 Screw Vial 1.5ml, 8-425 32x11.6mm,amber, 100 units</td>
			<td style="width:165px;">Thermo Fisher scientific</td>
			<td style="width:139px;">10560053</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X100 LOCK SKRUV VITT PTFE PACKNING 8-425 (Screw caps)</td>
			<td style="width:165px;">Thermo Fisher scientific</td>
			<td style="width:139px;">12458636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProteoMass LTQ/FT-Hybrid ESI Pos. Mode Cal Mix</td>
			<td style="width:165px;">Sigma-Aldrich</td>
			<td style="width:139px;">MSCAL5</td>
			<td>Calibration kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SNAKESKIN 10K MWCO&#160;</td>
			<td style="width:165px;">Thermo Fisher scientific</td>
			<td style="width:139px;">88245</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mathematica v.10&#160;</td>
			<td style="width:165px;">Wolfram Research</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a method for measuring metabolism in sorted subpopulations of complex cell communities using stable isotope tracing

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.

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...

Watch this Scientific Journal Video about A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing at JoVE.com

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

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.

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

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