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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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+heterogeneity.">Tumor heterogeneity.</a> Cancer Res. 44 (6), 2259-2265 (1984).</li><li>Prat, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cell+lines+derived+from+breast+cancers+and+normal+mammary+tissues+for+the+study+of+the+intrinsic+molecular+subtypes.">Characterization of cell lines derived from breast cancers and normal mammary tissues for the study of the intrinsic molecular subtypes.</a> Breast Cancer Res. Treat. 142 (2), 237-255 (2013).</li><li>Magistretti, P. 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L., Sivridis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+metabolic+pathways+between+cancer+cells+and+stromal+cells+in+colorectal+carcinomas:+A+metabolic+survival+role+for+tumor-associated+stroma.">Comparison of metabolic pathways between cancer cells and stromal cells in colorectal carcinomas: A metabolic survival role for tumor-associated stroma.</a> Cancer Res. 66 (2), 632-637 (2006).</li><li>Hollenbaugh, J. A., Munger, J., Kim, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolite+profiles+of+human+immunodeficiency+virus+infected+CD4++T+cells+and+macrophages+using+LC-MS/MS+analysis.">Metabolite profiles of human immunodeficiency virus infected CD4+ T cells and macrophages using LC-MS/MS analysis.</a> Virology. 415 (2), 153-159 (2011).</li><li>Richardson, G. 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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.

<table border="0" cellpadding="0" cellspacing="0" width="1001">
	<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. ...

The overall goal of this method is to use isotope tracing to measure the metabolism of sorted populations of complex cell communities. So with this method, we can study the metabolic differences between subpopulations of complex cell communities, and also the metabolic collaboration between those subpopulations. The main advantage with this technique is that by combining isotope tracing with cell sorting, we can get a more reliable measure of metabolism, and also we can validate how reliable the method is.Visual demonstration of this method is critical, as it combine different techniques, and the way cells are handled and the length of the procedure might affect cell metabolism. For metabolite extraction from a dish, first culture cells in a six-well plate in stable isotope labeled culture medium containing dialyzed supplements. When the cells reach 75%confluency, rinse the wells two times with 500 microliters of cold HBSS containing uniformly labeled glucose.Then add 600 microliters of 100%dry ice precooled methanol to each well, and transfer the plate to the dry ice. Use a cell scraper to detach the cells and then carefully transfer the extracts from each well into individual microcentrifuge tubes. Store cells at minus 80 degrees Celsius until mass spectrometry analysis.For metabolite extraction of sorted cells, culture cells in a 100 millimeter dish in labeled culture medium with dialyzed supplements, so that they reach 75%confluency by the sorting day. For pulse labeling of cell cycle sorted cells, culture the cells in unlabeled medium containing dialyzed supplements until they reach 75%confluency. Then, pulse label the cells for two hours by replacing the medium with fresh labeled culture medium.Rinse the dish with warm HBSS containing uniformly labeled glucose, and detach the cells with 1.5 milliliters of trypsin EDTA at 37 degrees Celsius. After four minutes, deactivate the trypsin with three milliliters of ice cold HBSS containing labeled glucose and dialyzed supplements, and transfer the cells to a 15 milliliter conical tube for centrifugation. Resuspend the pellet in HBSS containing labeled glucose, dialyzed supplements, and EDTA at a one to two times 10 to the six cells per milliliter concentration, and filter the cells through a 40-micron strainer to obtain a single cell suspension.Transfer the cells into a five milliliter tube and sort them on a flow cytometer. For metabolite extraction from mock sorted cells, gait out the debris and sort only the singlets. For metabolite extraction from cell cycle sorted cells, gait out the debris and doublets, and sort cells into G1 and SG2M, based on the geminin probe fluorescence.At the end of the sort, collect the cells by centrifugation and resuspend the pellet in 50 microliters of ice cold distilled water to obtain a homogeneous pellet. Then quickly extract the metabolites by adding 540 microliters of dry ice cooled methanol. Store the sample at minus 80 degrees Celsius until LCMS analysis.The extraction of metabolites from sorted cells must be well planned in advance, with experimental work being completed as quickly as possible to minimize the metabolic alterations. For analysis of the extracted metabolites, first select a number of metabolites with corresponding available standards that exhibit good quality sample peaks. Next, verify the quality of the peaks, taking care not to include false isotopes.Calculate the mass isotope from our fractions for the isotope labeled samples by dividing the peak area of each mass isotope from our fraction by the total peak areas of all the mass isotopes from our fractions. Then, calculate the labeled carbon fractions for the enrichment of carbon-13 using the formula for carbon enrichment. Finally, calculate the labeled nitrogen fractions for the enrichment of nitrogen-15 using the formula for nitrogen enrichment.In this representative experiment, 66 metabolites selected from the data analysis were present in the mock sorted cells, 60 of which appeared to be labeled with similar mass isotope from our distributions between the dish extracts and the mock sorted cells. For example, the mass isotope from our distribution of glutamate is similar between the dish and mock sorted extracts, indicating that the glutamate mass isotope from our distribution is also reliable in sorted cell populations. Analysis of HELA cells sorted into either G0, G1, or SG2M cell cycle stages using the FUCCI geminin probe reveals cytidine labeling in both populations with a higher enrichment in the SG2M stage, consistent with an increased de novo synthesis of nucleotides during the S phase.In this experiment, the mass isotope from our distribution exhibited the same pattern in both of the cell cycle sorted fractions, suggesting that the same synthesis pathway is used, but is more active in the SG2M stage, demonstrating that metabolic differences are readily detectable by this method, even between closely related subpopulations. After watching this video, you should have a good grasp on how to extract metabolites from sorted fractions, and understand the caveats. While attempting this procedure, it's important to remember to handle the cells quickly during sorting and metabolite extraction, as the manner and the length of time cells are handled can affect their metabolism.Once mastered, this technique can be performed in five hours. This procedure can be applied to other cell types. For example, immune cell subsets in blood, or different types of cells within a tumor, using a variety of probes, antibodies, or staining methods for sorting.

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Research

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Biochemistry

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.

Here, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff-base complex. Hydrolysis conditions presented here are suitable for use when retention of acid-labile side-chain protecting groups is required. This technique may be adaptable to a variety of unnatural amino acid substrates.

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). SM is a common sphingolipid composed of a phosphorylcholine and a ceramide as the hydrophilic and hydrophobic component, respectively. A number of SM species are present in mammalian cells due to a variety in the sphingoid long chain base (LCB) and an N-acyl moiety in the ceramide. In this report, we show a method of estimating the number of carbon and double bonds in a LCB and an N-acyl moiety based on their corresponding product ions in MS/MS/MS (MS3) experiments. In addition, we present a quantitative analysis method for SM using two stable isotopically labeled SM species, which facilitates determining the range used in SM quantitation. The present method will be useful in characterizing a variety of SM species in biological samples and industrial products such as cosmetics.

Here, we present a protocol to quantify and qualify each sphingomyelin species using multiple reaction monitoring and MS/MS/MS mode, respectively.

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and paleoenvironmental research from recent historical periods back to over 10 million years ago. Bulk approaches provide a representative sample for the period of enamel mineralization, while sequential samples within a tooth can track dietary and environmental changes during this period. While these methodologies have been widely applied and described in archaeology, ecology, and paleontology, there have been no explicit guidelines to aid in the selection of necessary lab equipment and to thoroughly describe detailed laboratory sampling and protocols. In this article, we document textually and visually, the entire process from sampling through pretreatment and diagenetic screening to make the methodology more widely available to researchers considering its application in a variety of laboratory settings.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been used as a proxy for individual diet and environmental reconstruction. Here, we provide a detailed description and visual documentation of bulk and sequential tooth enamel sampling as well as pretreatment of archaeological and paleontological samples.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1&#946;-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC. 
Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.

Here, an experimental workflow is presented that enables the detection of caspase-8 processing directly at the death-inducing signaling complex (DISC) and determines the composition of this complex. This methodology has broad applications, from unraveling the molecular mechanisms of cell death pathways to the dynamic modeling of apoptosis networks.

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ...