HAC was the Milton E. Cassel scholar of the Rita Allen Foundation (http://www.ritaallenfoundation.org). This work was funded by grants to HAC from National Institute of General Medical Sciences R01 GM081686, R01 AR070245, National Institute of General Medical Sciences R01 GM0866465, the Eli &#38; Edythe Broad Center of Regenerative Medicine &#38; Stem Cell Research (Rose Hills and Hal Gaba awards), the Iris Cantor Women&#8217;s Health Center/UCLA CTSI NIH Grant UL1TR000124, the Leukemia Lymphoma Society, Impact awards from the Jonsson Comprehensive Cancer Center to WEL and HAC. Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number P50CA092131. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. HAC is a member of the Eli &#38; Edythe Broad Center of Regenerative Medicine &#38; Stem Cell Research, the UCLA Molecular Biology Institute, and the UCLA Bioinformatics Interdepartmental Program.

All experiments described were approved by the Animal Care Committee at the University of California, Los Angeles.
1. Generate Slides of Frozen Mouse Skin
<ol>
	<li>Euthanize mice in accordance with institution policy. 
	Note: Follow institutional policy for protective clothing. All protocols involving animals must be approved by the Institutional Animal Care Committee.</li>
	<li>Remove the mouse&#8217;s hair with an animal hair trimmer.</li>
	<li>Make incisions in the skin using scissors. Using forceps, lift the skin away from the mouse. Using scissors, cut the skin section away.</li>
	<li>Fill the cryomold with freezing reagent compound (see Table of Materials).</li>
	<li>Place skin sections into filled cryomolds using needle-nosed forceps. Orient skin so that tissue slices will generate a cross section of the skin from the epidermis to the dermis, hypodermis and muscle (Figure 2). 
	Note: If possible, mount experimental and control mice together in the same cryomold so they can be sectioned onto the same slide and processed together. Take care not to create bubbles.</li>
	<li>Place cryomold on the flat surface of a block of dry ice in an ice bucket and let freeze. Continue to observe the orientation of the skin. Adjust if necessary. 
	Note: Wear cryogenic gloves when handling dry ice.</li>
	<li>Transfer cryomolds into a -80 &#176;C freezer for storage. Samples can be maintained in the freezer for approximately 3 months without significant loss of enzymatic activity. Do not let frozen sections dry out.</li>
	<li>Using a cryostat at -20 &#176;C, slice tissue to create sections 7 - 10 &#181;m thick for the best skin morphology21.</li>
</ol>
2. Preparing Slides for Staining
<ol>
	<li>Establish the set of slides to be tested. Include a control slide on which NAD will be withheld and another control slide on which the substrate, lactate, will be withheld. Include a positive control slide from a frozen tissue block previously investigated.</li>
	<li>Briefly fix slides containing skin sections with 4% formalin for 5 min either by pipetting 1 mL of 4% formalin onto the slide, or when processing multiple slides, by dipping slides into a container containing 4% formalin. 
	Note: The fixation will ensure the skin does not peel off from the slides during the following procedures and preserve the skin morphology. 
	Caution: Formalin is a carcinogen and hazardous; wear gloves, do not let it touch your skin and perform this step in a chemical fume hood.</li>
	<li>Wash slides with at least 1 mL phosphate buffered saline, pH 7.4, per slide either by pipetting or dipping.</li>
</ol>
3. Preparing Staining Solution
<ol>
	<li>Vortex together reagents for lactate dehydrogenase activity staining (50 mM Tris pH 7.4, 750 &#181;M NADP, 80 &#181;M phenazine methosulfate (PMS), 600 &#181;M nitrotetrazolium blue chloride, and 30 mM lactate) in an appropriately sized tube depending on the amount of staining solution required. 
	Note: 1 mL is sufficient to completely cover one slide.</li>
	<li>Prepare a second solution in which all reagents are present except NAD. Prepare a third solution in which all reagents are present except lactate.</li>
</ol>
4. Incubate Slides in Staining Solution
<ol>
	<li>Gently pipet the staining solution or control solutions onto the prepared slides covering the section in its entirety (1 mL is sufficient for one slide). If multiple slides are being processed together, dip all slides into the staining solution at the same time (make sure the container has enough solution to completely cover the tissue section).</li>
	<li>Incubate the slides in a humidified environment at 37 &#176;C in the dark. If staining solution is on top of the slide, place the slide in a humidified environment to prevent evaporation.</li>
</ol>
5. Monitor the Slides
<ol>
	<li>Monitor the conversion of the staining solution from clear to blue by visual inspection. When the samples have reached the desired level of blueness, stop the reaction by removing the staining solution. 
	Note: For skin, 10 min is sufficient. The time will depend on the organ and the enzymatic activity.</li>
	<li>Rinse slides with phosphate buffered saline by pipetting (1 mL is sufficient for each slide) or dipping. 
	Note: Any samples that will be compared to each other must be maintained in staining solution for the same amount of time.</li>
</ol>
6. Counterstain and mount
<ol>
	<li>Pipet a counterstain onto the slides when the slides have reached an appropriate level of blueness. 
	Note: Counterstains that turn the nuclei red or green will provide good contrast with the blue created by the formazan precipitant.</li>
	<li>Mount with aqueous or non-aqueous mounting medium22. One to three drops (40 - 50 &#181;L) of mounting medium is sufficient depending on the size of the cover slip.</li>
</ol>
7. Image slides and quantify
<ol>
	<li>Image slides under a light microscope. Take photographs of experimental and control samples and all negative controls at 10X, 20X, and 40X magnification.</li>
	<li>Determine the intensity of blue stain in different regions with image analysis software.</li>
</ol>

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Enzyme Histochemistry: A Laboratory Manual of Current Methods. , (1992).</li><li>Winzer, K., Van Noorden, C. J., Kohler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+cytochemical+analysis+of+glucose-6-phosphate+dehydrogenase+activity+in+living+isolated+hepatocytes+of+European+flounder+for+rapid+analysis+of+xenobiotic+effects.">Quantitative cytochemical analysis of glucose-6-phosphate dehydrogenase activity in living isolated hepatocytes of European flounder for rapid analysis of xenobiotic effects.</a> J Histochem Cytochem. 49, 1025-1032 (2001).</li><li>Negi, D. S., Stephens, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+the+histochemical+localization+of+glucose-6-phoshate+dehydrogenase+in+animal+and+plant+tissues.">An improved method for the histochemical localization of glucose-6-phoshate dehydrogenase in animal and plant tissues.</a> J Histochem Cytochem. 25, 149-154 (1977).</li><li>Boren, J., 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=In+situ+localization+of+transketolase+activity+in+epithelial+cells+of+different+rat+tissues+and+subcellularly+in+liver+parenchymal+cells.">In situ localization of transketolase activity in epithelial cells of different rat tissues and subcellularly in liver parenchymal cells.</a> J Histochem Cytochem. 54, 191-199 (2006).</li><li>Miller, 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=Exploring+metabolic+configurations+of+single+cells+within+complex+tissue+microenvironments.">Exploring metabolic configurations of single cells within complex tissue microenvironments.</a> Cell Metab. , (2017).</li><li>Shen, J., 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=Prognostic+value+of+serum+lactate+dehydrogenase+in+renal+cell+carcinoma:+a+systematic+review+and+meta-analysis.">Prognostic value of serum lactate dehydrogenase in renal cell carcinoma: a systematic review and meta-analysis.</a> PLoS One. 11, e0166482 (2016).</li><li>Zhang, X., 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=Prognostic+significance+of+serum+LDH+in+small+cell+lung+cancer:+A+systematic+review+with+meta-analysis.">Prognostic significance of serum LDH in small cell lung cancer: A systematic review with meta-analysis.</a> Cancer Biomark. 16, 415-423 (2016).</li><li>Petrelli, F., 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=Prognostic+role+of+lactate+dehydrogenase+in+solid+tumors:+a+systematic+review+and+meta-analysis+of+76+studies.">Prognostic role of lactate dehydrogenase in solid tumors: a systematic review and meta-analysis of 76 studies.</a> Acta Oncol. 54, 961-970 (2015).</li><li>Valvona, C. J., Fillmore, H. L., Nunn, P. B., Pilkington, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+and+function+of+lactate+dehydrogenase+A:+Therapeutic+potential+in+brain+tumor.">The regulation and function of lactate dehydrogenase A: Therapeutic potential in brain tumor.</a> Brain Pathol. 26, 3-17 (2016).</li><li>Markert, C. L., Shaklee, J. B., Whitt, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+a+gene:+Multiple+genes+for+LDH+isozymes+provide+a+model+of+the+evolution+of+gene+structure,+function+and+regulation.">Evolution of a gene: Multiple genes for LDH isozymes provide a model of the evolution of gene structure, function and regulation.</a> Science. 189, 102-114 (1975).</li><li>Read, J. A., Winter, V. J., Eszes, C. M., Sessions, R. B., Brady, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+altered+activity+of+M-+and+H-isozyme+forms+of+human+lactate+dehydrogenase.">Structural basis for altered activity of M- and H-isozyme forms of human lactate dehydrogenase.</a> Proteins. 43, 175-185 (2001).</li><li>Holness, M. J., Sugden, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+pyruvate+dehydrogenase+complex+activity+by+reversible+phosphorylation.">Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation.</a> Biochem Soc Trans. 31, 1143-1151 (2003).</li><li>Yi, W., 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=Phosphofructokinase+1+glycosylation+regulates+cell+growth+and+metabolism.">Phosphofructokinase 1 glycosylation regulates cell growth and metabolism.</a> Science. 337, 975-980 (2012).</li><li>Fan, J., 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=Tyrosine+phosphorylation+of+lactate+dehydrogenase+A+is+important+for+NADH/NAD(+)+redox+homeostasis+in+cancer+cells.">Tyrosine phosphorylation of lactate dehydrogenase A is important for NADH/NAD(+) redox homeostasis in cancer cells.</a> Mol Cell Biol. 31, 4938-4950 (2011).</li><li>Zhao, D., 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=Lysine-5+acetylation+negatively+regulates+lactate+dehydrogenase+A+and+is+decreased+in+pancreatic+cancer.">Lysine-5 acetylation negatively regulates lactate dehydrogenase A and is decreased in pancreatic cancer.</a> Cancer Cell. 23, 464-476 (2013).</li><li>Vanderlinde, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+total+lactate+dehydrogenase+activity.">Measurement of total lactate dehydrogenase activity.</a> Ann Clin Lab Sci. 15, 13-31 (1985).</li><li>Nelson, S. J., 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=Metabolic+imaging+of+patients+with+prostate+cancer+using+hyperpolarized+[1-(1)(3)C]pyruvate.">Metabolic imaging of patients with prostate cancer using hyperpolarized [1-(1)(3)C]pyruvate.</a> Sci Transl Med. 5, 198ra108 (2013).</li><li>Flores, 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=Lactate+dehydrogenase+activity+drives+hair+follicle+stem+cell+activation.">Lactate dehydrogenase activity drives hair follicle stem cell activation.</a> Nat Cell Biol. , (2017).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryosectioning+tissues.">Cryosectioning tissues.</a> CSH Protoc. 2008, (2008).</li><li>Espada, J., 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=Non-aqueous+permanent+mounting+for+immunofluorescence+microscopy.">Non-aqueous permanent mounting for immunofluorescence microscopy.</a> Histochem Cell Biol. 123, 329-334 (2005).</li><li>Hisada, R., Yagi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1-Methoxy-5-methylphenazinium+methyl+sulfate.+A+photochemically+stable+electron+mediator+between+NADH+and+various+electron+acceptors.">1-Methoxy-5-methylphenazinium methyl sulfate. 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The authors have no competing interests to disclose.

The method described here can be used to monitor the activity of lactate dehydrogenase or other metabolic enzymes that generate NADH or NADPH, in different cell types within a tissue or within different portions of a tissue over time. Lactate dehydrogenase is an important enzyme for understanding the biology of stem cells and tumors, and the ability to monitor lactate dehydrogenase activity in individual cells is likely to provide important insights into the function of this enzyme.
One important advantage of this protocol compared with other methodologies is that the ability to monitor enzymatic activity, rather than just protein abundance, can result in more conclusive information about not just where lactate dehydrogenase enzymes are present, but where they are actually functional. The approach described here can be combined with immunohistochemistry so that the level of a selected protein, for instance, a cell lineage marker, is determined in the same tissue samples as lactate dehydrogenase activity. As compared with MRI-based approaches for monitoring lactate dehydrogenase activity, the protocol provided has the advantage that it can provide greater spatial resolution and help to determine the specific cells within a tissue that have high enzymatic activity. As compared with approaches to mapping metabolism based on fluorogenic substrates, detecting signal with the protocol described is not limited by autofluorescence1. Miller and co-authors performed a similar analysis of the time dependence and substrate dependence of five different enzymes by monitoring conversion to formazan7. The reactions were discovered to follow stoichiometric principles of enzyme kinetics. Miller and co-authors distinguished mitochondrial enzymes from non-mitochondrial enzymes by co-localization with mitochondrial staining7. They used 5-Cyano-2,3-di-(p-tolyl)tetrazolium chloride (CTC) as a detection reagent followed by confocal microscopy for subcellular co-localization analysis7.
The method described has disadvantages as well. While MRI-based approaches can be used in living organisms, the approach described requires frozen or fresh tissue. Frozen tissues are only suitable for a few months because the activity of the enzymes degrade over time, even when frozen. Further, because the substrate is provided in excess, the approach provides information on &#34;activity potential&#34; assuming substrate is present. If the levels of the substrate are limiting, then the activity distribution observed with this assay may deviate from the amount of activity that occurs physiologically. In addition, if there are different isoforms of the same enzyme that perform the same enzymatic activity, the method presented here is not able to distinguish between them. For instance, the method presented cannot distinguish between the activity of LDHA versus LDHB. One approach to address this issue would be to use genetically engineered mice in which a single enzyme isoform is genetically inactivated.
We have experimented with PMS, a photochemically stable electron carrier that mediates electron transfer between NADH and tetrazolium dyes23. While some scientists have reported that the PMS inhibits glucose 6-phosphate dehydrogenase activity and found PMS unhelpful5, we found the PMS to be valuable for enzyme localization and include it in our protocol. Some other authors have reported using polyvinyl alcohol to limit diffusion of glucose 6-phosphate dehydrogenase enzyme1. In our experiments, we did not find the polyvinyl alcohol to be helpful and eliminated it.
A few steps are critical for the success of the staining. One important issue is ensuring that the tissue samples are placed properly in the cyromolds. Skin sections should be laid flat so that slices cut across the skin. In addition, if multiple sections are to be prepared, it is ideal if they are embedded within the same cryomold so they can be sliced together on the same slide. This will help to eliminate variation due to the thickness of the slide that is cut with the cryostat. It is also important to ensure that the tissue still retains enzymatic activity.
The best time to end the reaction should be determined empirically. It is necessary to monitor the slides closely during the incubation period and determine the appropriate time to add counterstain and mount the slides based on the intensity of the blue stain. If the samples are too lightly stained, it will not be possible to determine where the activity is. If the samples are overstained, the formazan precipitate can spread, making it difficult to ascertain the specific areas that were originally high in activity.
We anticipate this protocol will be valuable for scientists with a wide range of questions about the role of metabolism. Applications include co-staining for metabolic activity and cell lineage markers or proliferation markers, assessing the contributions of different enzymes with genetic models, assessing subcellular localization of enzymatic activity, monitoring enzymatic activity in diseased tissue or during development.

Understanding the locations within tissues in which enzymes have high or low activity is essential for understanding development and physiology. Transcript or protein levels are often used as surrogates for enzymatic activity. While such studies can be informative, they do not provide information that can be critical for determining an enzyme&#39;s activity, such as post-translational modifications, the presence of protein complexes, or the enzyme&#39;s subcellular localization. When enzymatic activity is directly measured, it is often monitored in homogenized protein lysates that no longer contain information about individual cells within the mixture or the spatial distribution of the cells with high or low activity within a tissue.
We provide here a detailed protocol for mapping the spatial distribution of enzymatic activity within a tissue sample. The methodology is based on earlier studies demonstrating that tetrazolium salts can be used to localize the activity of dehydrogenases, reductases, and oxidases in frozen tissue1. With these methods, a water-insoluble formazan is formed when protons are transferred to a tetrazolium salt2,3. Glucose-6-phosphate dehydrogenase generates NADPH and a proton, and has been detected with tetrazolium activity. Glucose-6-phosphate dehydrogenase has been monitored in in European flounder hepatocytes4, in alveolar type 2 cells of the lungs5 and nephrons of the kidney5. Tetrazolium salts have also been used to monitor transketolase activity in frozen tissue6. A similar approach was recently&#160;used to monitor the activity of multiple dehydrogenases in the same tissue on adjacent slides7.
We describe here a method to use tetrazolium salts to monitor the spatial distribution of lactate dehydrogenase activity (Figure 1). Lactate dehydrogenase can convert pyruvate generated by glycolysis to lactate, and the reverse reaction. Lactate dehydrogenase activity is consequently an important determinant of pyruvate&#39;s entrance into the tricarboxylic acid cycle versus its secretion as lactate. Lactate levels in the blood are often used to diagnose a range of diseases, including cancer8,9,10, because it can signal that illness or injury has damaged cells and the enzyme has been released.
There are four lactate dehydrogenase genes: LDHA, LDHB, LDHC, and LDHD11. LDHA and LDHB are thought to have arisen from duplication of an early LDHA gene12. LDH is active as a tetramer and LDHA and LDHB can form homotetramers and heterotetramers with each other. LDHA is reported to have higher affinity for pyruvate, while LDHB is reported to have higher affinity for lactate, and to preferentially convert lactate to pyruvate13. The LDHA promoter contains binding sites for the HIF1&#945;, cMYC and FOXM1 transcription factors11. In addition, like many other glycolytic enzymes14,15, LDH can be modified by post-translational modifications. Fibroblast growth factor receptor 1 can phosphorylate LDHA at Y10, which promotes tetramer formation, or Y83, which promotes NADH substrate binding16. LDHA can also be acetylated17. For these reasons, a complete understanding of LDH activity requires monitoring not only LDH protein levels, but the enzyme&#39;s activity as well.
In addition to the method we present here, other approaches have been used to monitor lactate dehydrogenase activity. Lactate dehydrogenase activity can be monitored spectrophotometrically in homogenized protein lysate. The generation of NADH as lactate is converted to pyruvate can be measured based on absorbance at 340 nm, while the disappearance of NADH can be monitored as pyruvate is converted to lactate18. Lactate dehydrogenase activity has also been monitored with magnetic resonance imaging (MRI). 13C- pyruvate can be administered and the conversion of pyruvate to lactate can be monitored as the ratio of [1-13C]lactate/[1-13C]pyruvate. Elevated ratios of [1-13C]lactate/[1-13C]pyruvate have been observed in cancer tissue19. While MRI-based approaches can provide information on lactate dehydrogenase activity in normal and disease tissues, the methodologies do not have the resolution needed to determine the activity level in specific cells. The methodology provided here can provide information on lactate dehydrogenase activity in tissue areas and even in single cells.
Using in situ activity assays, we found that the activity of lactate dehydrogenase is high in the hair follicle stem cells of mouse skin20. We also used the method to monitor the activity of lactate dehydrogenase in cultured embryonic stem cells and found the activity is higher in the stem cells than the feeder layer. Finally, we monitored the activity of lactate dehydrogenase in fresh mouse aorta and observed staining in smooth muscle cells. We describe here a detailed protocol for monitoring lactate dehydrogenase activity in frozen mouse skin.

<table border="0" cellpadding="0" cellspacing="0" style="width:815px;" width="814">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:312px;">Surgical instruments</td>
			<td style="width:164px;"></td>
			<td style="width:121px;"></td>
			<td style="width:217px;">For collecting skin from euthanized mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Tissue-tek cryomold 25 mm x 20 mm x 5 mm</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">NC9511236</td>
			<td style="width:217px;">For freezing mouse skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Tissue-Tek O.C.T. compound</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">NC9638938</td>
			<td style="width:217px;">For mounting cryomolds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Ice bucket</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">07-210-106</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Dry Ice</td>
			<td style="width:164px;"></td>
			<td style="width:121px;"></td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Polysine Adhesion Slide</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">12-545-78</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">4% formalin</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">23-245-684</td>
			<td style="width:217px;">Dilluted in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">phosphate buffered saline, pH 7.4</td>
			<td style="width:164px;"></td>
			<td style="width:121px;"></td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">vortex</td>
			<td style="width:164px;"></td>
			<td style="width:121px;"></td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Tris base</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">23-245-684</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">NAD</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:121px;">N7004</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Phenazine methosulfate</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:121px;">P9625</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:312px;">Nitrotetrazolium blue chloride</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:121px;">N6876</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Lithium L-lactate</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:121px;">L2250</td>
			<td style="width:217px;">Substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">37&#176;C incubator (or tissue culture incubator)</td>
			<td style="width:164px;"></td>
			<td style="width:121px;"></td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Braziliant! Counter stain</td>
			<td style="width:164px;">Anatech</td>
			<td style="width:121px;">861</td>
			<td style="width:217px;">Counter stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Mounting medium</td>
			<td style="width:164px;">Vector Laboratories</td>
			<td style="width:121px;">H-5000&#160;</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Cover slips for slides</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:121px;">12-544D</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Light microscope</td>
			<td style="width:164px;"></td>
			<td style="width:121px;"></td>
			<td style="width:217px;"></td>
		</tr>
	</tbody>
</table>

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 have previously reported results for in situ activity assays in mouse skin20. As shown in Figure 3, we observed high levels of lactate dehydrogenase activity in the hair follicle stem cells at the base of the hair follicle when the procedures described above were followed. These findings were corroborated by fluorescence activated cell sorting of skin for hair follicle stem cells and confirming high lactate dehydrogenase activity in the stem cell compartment with activity assays on sorted cells.
Based on our findings that the hair follicle stem cells have high lactate dehydrogenase activity, we extended our studies of cultured embryonic stem cells. Mouse embryonic fibroblasts serving as a feeder layer were analyzed alone or in the presence of embryonic stem cells. To analyze cultured cells, the medium was aspirated, and the cells were briefly fixed and incubated with lactate dehydrogenase staining solution as described above. We observed high lactate dehydrogenase activity in the embryonic stem cells as compared with the mouse embryonic fibroblasts (Figure 4).
We have also applied the staining protocol to freshly isolated tissue. Mouse aortas were dissected and sliced open. The vessels were opened and immobilized so that the inside lumen of the aortas was accessible. Samples were incubated with Hank&#39;s Balanced Salt Solution containing 0.5% Triton-X for 10 min, then with the lactate dehydrogenase staining solution for 10 min. Lactate dehydrogenase staining solution was applied to the fresh tissue. After incubation, images were collected, showing staining in the smooth muscle cells (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57760/57760fig1.jpg" /> 
Figure 1: Chemical activity of lactate dehydrogenase and its detection. Lactate dehydrogenase converts lactate + NAD to pyruvate + NADH + H+. The proton that is generated will reduce nitroblue tetrazolium to formazan, which will form a blue precipitate. <a href="/files/ftp_upload/57760/57760fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57760/57760fig2.jpg" /> 
Figure 2: Schematic of orienting mouse skin in cryomolds. Mouse skin should be oriented within cryomolds so that each cut section will contain a cross section of the entire skin from the epidermis, dermis, hypodermis, and muscle. <a href="/files/ftp_upload/57760/57760fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57760/57760fig3.jpg" /> 
Figure 3: Lactate dehydrogenase staining of mouse skin reveals high activity in hair follicle stem cells. Mouse skin was frozen in cryomolds and incubated with lactate dehydrogenase staining solution contain lactate. Lactate dehydrogenase activity was prominent in hair follicle stem cells. (A, B) Two images of lactate dehydrogenase activity assay of mouse skin. (C, D) Two images of control staining with the substrate lactate withheld. Scale bar = 50 &#181;m. <a href="/files/ftp_upload/57760/57760fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57760/57760fig4.jpg" /> 
Figure 4: High lactate dehydrogenase activity in cultured mouse embryonic stem cells. Cultured mouse embryonic stem cells grown on an irradiated mouse embryonic fibroblast feeder layer were analyzed for lactate dehydrogenase activity. High activity was observed in the stem cells compared with the fibroblasts. Mouse embryonic fibroblasts showed similar levels of staining with and without irradiation. Images were taken with a 20X objective. Scale bar = 50 &#181;m. (A, B, D, E) Mouse embryonic fibroblasts and stem cells. (C, F) Mouse embryonic fibroblasts alone. (A-C) Images were taken after 7 minutes of incubation with staining solution. (D-F) Images were taken after 20 minutes of incubation with staining solution. <a href="/files/ftp_upload/57760/57760fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57760/57760fig5.jpg" /> 
Figure 5: High lactate dehydrogenase activity in the smooth muscle cells surrounding the mouse aorta. Aortas were isolated from euthanized mice. Vessels were fixed for 2 minutes in 2% paraformaldehyde and washed 3 x 10 minutes in 1x phosphate buffered saline. Vessels were incubated with Hank&#39;s balanced salt solution with 0.5% Triton-X and washed 3 x 5 minutes in phosphate buffered saline. Lactate dehydrogenase staining solution was added and samples were incubated for 10 min at 37&#176;C and washed 3 x 5 min with 1x phosphate-buffered saline. Samples were then fixed for 1 hour in 2% paraformaldehyde, washed 3 x 5 min with 1x phosphate-buffered saline and imaged. (A) Lactate dehydrogenase solution containing lactate. (B) Lactate dehydrogenase solution without lactate as a negative control. For each section, there is a lower magnification image on the left and a designated area is shown with higher magnification on the right. (A, B) Complete lactate dehydrogenase solution containing lactate was used for staining. B is a magnified image of the indicated area in image A. (C, D) Lactate dehydrogenase solution without lactate was used for staining as a negative control. D is a magnified image of the indicated area in C. A and C were taken with 20X objectives (scale bar = 50 &#181;m) and B and D were taken with a 40X objective (scale bar = 20 &#181;m). <a href="/files/ftp_upload/57760/57760fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue at JoVE.com

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in 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 ...

mapping-metabolism-monitoring-lactate-dehydrogenase-activity-directly

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.3K Views.  UCLA. 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.

Video: Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

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.

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. ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

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 ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest.&#160;Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 &#176;C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

The purity and integrity of the isolated RNA is a vital step in RNA dependent assays. Here, we present a practical, rapid, and inexpensive method to extract RNA from a small quantity of undamaged pancreatic tissue.

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...

Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains of &#945;1,4-linked glucose residues with branches introduced through the addition of &#945;1,6-linkages. Understanding how the synthesis and degradation of glycogen are regulated and how glycogen attains its characteristic branched structure requires the study of the enzymes of glycogen storage. However, the methods most commonly used to study these enzyme activities typically employ reagents or techniques that are not available to all investigators. Here, we discuss a battery of procedures that are technically simple, cost-effective, and yet still capable of providing valuable insight into the control of glycogen storage. The techniques require access to a spectrophotometer, operating in the range of 330 to 800 nm, and are described assuming that the users will employ disposable, plastic cuvettes. However, the procedures are readily scalable and can be modified for use in a microplate reader, allowing highly parallel analysis.

Techniques to measure the activity of key enzymes of glycogen metabolism are presented, using a simple spectrophotometer operating in the visible range.

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains ...