The HCC4017 and HBEC30KT cell lines were kindly provided by Dr. John Minna&#8217;s lab. This work was supported by the Cecil H. and Ida Green funds to Dr. Li Zhang.

CAUTION: While working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Dispose off all waste following local radiation safety guidelines.
1. Preparation of Cells
<ol>
	<li>Seed cells in 3.5 cm plates such that the confluency reaches 80%-90% on the day of assay.
	<ol>
		<li>Note that seeding confluency for cells depends upon the cell type and their growth rate. When treating cells with a reagent for a certain number of days, seed cells so that the confluency is 80%-90% on the day of heme measurement. If the number of cells obtained from 3.5 cm plates is insufficient, use 6 cm plates with same amount of radioactive ALA as in the next step. 
		Note: Use of individual plates is recommended because it is easier to handle individual plates.</li>
	</ol>
	</li>
	<li>A day before heme measurement, add 0.1-0.3 &#181;Ci [14C] 5-ALA and cold ALA to reach a final concentration of 20 &#181;M of total ALA to each culture dish in a work area fit for working with radioactive materials and put the plates back into the incubator. Ideally, incubate with radioactive ALA for 16 hr. 
	Note: Radiolabeled 5-ALA required may vary between cell lines. A radioactive count per minute (cpm) of at least 100 is required to minimize the error associated with counting. It is advisable to determine a minimum amount of [14C] 5-ALA required for the cell lines under study. For most experiments 0.1-0.3 &#181;Ci is sufficient to get a good measurement. Addition of cold ALA is optional.</li>
</ol>
2. Heme Extraction
<ol>
	<li>Place the cell culture dishes on ice and take them to the area fit for radioactivity work.
	<ol>
		<li>Perform the whole extraction and evaporation process in an explosion-proof hood. Because the stored ether can explode, use the ether in small bottles and evaporate any remaining liquids, do not store ether in an open bottle for more than one month. Do not use acetone near open flames and heaters as it is highly flammable. Store acetone containing reagents in glass bottles as it dissolves tissue culture plastics.</li>
	</ol>
	</li>
	<li>Aspirate off the medium using a pipette. Wash cells one time with 1 ml of 1x cold PBS. Aspirate off PBS.</li>
	<li>Add 1 ml of 1x cold PBS to the plates and collect the cells at the bottom of the plate using a rubber policeman. Transfer the collected cells from each plate to a labeled pre-chilled 2 ml tube. Collect all cells in 2 ml tubes. 
	Note: Use a fresh rubber policeman for each plate for scraping cells from each plate. For each plate you would require four 2 ml tubes, a 1.5 ml tube and a scintillation vial.</li>
	<li>Spin at 15,000 x g for 1 min at 4 &#176;C.</li>
	<li>Take out PBS using a pipette, give a short spin and remove the residual PBS. Resuspend cells in 100 &#956;l of 1x PBS. Vortex to resuspend cells and keep on ice.</li>
	<li>Take 10 &#956;l from step 5 and transfer to a 1.5 ml microcentrifuge tube and store on ice to measure protein concentration. 
	Note: Cell lysis buffer should be added to the cells and kept on ice to measure protein concentration later.</li>
	<li>Spin the remaining 90 &#181;l from step 5 at 5,000 x g for 30 sec at 4 &#176;C; remove PBS completely using a 200 &#181;l pipette tip.</li>
	<li>Add 300 &#181;l of heme extraction buffer (see Materials List) to each tube. Then vortex for 15 sec to mix and put on ice for 1 min, repeat this 3 times.
	<ol>
		<li>Add heme extraction buffer (HEB) and vortex the tube quickly to resuspend the pellet, do only one or two tubes at a time because the pellet should be resuspended quickly without being left in the HEB for too long. Once HEB is added to all the tubes and pellet is resuspended then follow the 15 sec vortex and 1 min on ice cycle as in step 8. Note that pellet may not go completely into the HEB if the pellet is left in the HEB buffer for a longer time and not resuspended quickly.</li>
	</ol>
	</li>
	<li>Add 1.2 ml of diethyl ether to each tube and vortex for 30 sec. Spin at 15,000 x g for 5 min at 4 &#176;C. 
	Note: Diethyl ether runs out of pipette tips quickly. To control this, pipette diethyl ether up and down once or twice, doing so helps hold the diethyl ether in the pipette. Repeat this every time a fresh pipette tip is used.</li>
	<li>Transfer top (ether) phase with 1 ml pipette to a new microcentrifuge tube. Add 0.5 ml of 2 N HCl, vortex to mix and spin as in step 9. 
	Note: It is difficult to see the separation between ether and HCl phases. Therefore, add small amounts of Trypan blue in 2 N HCl solution to be used during the experiment, enough to give it a pale blue color. This will help distinguish the two phases. The lower phase will be blue and the upper ether phase will be colorless. Transfer the upper colorless ether phase to a new tube and discard the lower HCl (blue) phase.</li>
	<li>Repeat step 10 two more times to wash the ether phase with 2 N HCl. Transfer only the top ether phase to a new tube during each repetition and discard the lower HCl phase.</li>
	<li>After the third wash with HCl, transfer the top phase with 1 ml pipette to a scintillation vial. Dry the samples by evaporating the diethyl ether by keeping the samples in the chemical hood O/N.</li>
</ol>
3. Measurement of Radioactivity
<ol>
	<li>Next day, add 5 ml of scintillation fluid to the dried samples from step 2.12.</li>
	<li>Shake well to mix. Measure the radioactivity by using a scintillation counter.</li>
</ol>
4. Calculations
<ol>
	<li>Lyse the cells from step 2.6 using equal amounts of CelLytic M buffer (see Materials List) and spin at 14,000 x g for 5 min at 4 &#176;C.</li>
	<li>Take an aliquot of the supernatant and measure the protein concentration using BCA protein assay kit. 
	Total amount of protein in mg (TP) = (90 &#181;l * conc. of protein in &#181;g/&#181;l) / 1,000 
	Heme synthesis level (radioactivity/TP) = pmol/mg of protein.</li>
</ol>

We have nothing to disclose.

Heme plays a key role in the generation of cellular energy via respiration26. Altered heme metabolism is known to be associated with various diseases including cancer28,41. Inhibition of heme synthesis is known to cause cell cycle arrest and apoptosis in Hela cells26,41. It has been shown that high heme synthesis level is associated with the progression of lung cancer cells28. Therefore, it would be of great importance to measure the levels of heme biosynthesis in cells under d...

Heme, a complex of ferrous iron and protoporphyrin IX is a central molecule for transporting and utilizing oxygen in virtually all living organisms1-3. The unique structure of heme enables it to function as a carrier of diatomic gases and electrons, as well as to perform various other functions1-5. For example, heme binds to oxygen in hemoglobin and myoglobin for the transfer and storage of oxygen6,7. It also works as an electron carrier in the cytochromes during respiration and acts as an electron donor for redox reactions catalyzed by cytochrome P450 enzymes8,9. One of the most significant features of heme is that, it can play regulatory roles in cellular and molecular processes such as gene transcription, protein synthesis and micro-RNA biogenesis4. For example, it affects the transcription of many genes by controlling the activity of mammalian transcriptional repressor Bach1 and the mammalian nuclear receptor Rev-erb&#945;10-15. Heme regulates the activation of heme activator protein (Hap) 1 which plays an important role in the activation of genes involved in respiration and controlling oxidative damage, in response to heme or oxygen16. Heme also regulates gene transcription in neuronal cells via nerve growth factor (NGF) signaling3,17-20. It also regulates protein synthesis in mammalian erythroid cells by modulating the activity of heme-regulated eIF2&#945; kinase (HRI)21-24. Furthermore, heme affects the activity of key signaling proteins such as tyrosine kinase Jak2 and Src, which are essential for proper cell functioning and cell growth4,20,25. It was found that in HeLa cells heme inhibition causes cell cycle arrest and activation of markers associated with senescence and apoptosis26. Both Heme deficiency or increased levels of heme are associated with severe health effects in humans27. Recent molecular and epidemiological studies have shown a positive association of high heme intake and increased risk of diseases, such as type-2 diabetes, coronary heart disease and several cancers including lung cancer, colorectal cancer and pancreatic cancer27,28. Using a matched pair of normal and cancer lung cells authors&#39; lab have found that cancer cells have increased levels of oxygen consumption, heme synthesis and proteins involved in heme uptake and oxygen utilization28. Interestingly, inhibition of heme synthesis decreased oxygen consumption, proliferation, migration and colony formation of cancer cells28. Thus, the fluctuation in the levels of endogenous heme plays an important role in the regulation of molecular and cellular processes3,4,28,29.
In mammals, the biosynthesis of heme occurs in eight steps, involving enzymes located in the mitochondria and the cytosol4 (Figure 1). Heme biosynthesis begins in the matrix of mitochondria with the condensation of glycine and succinyl-coA to form 5-aminolevulinic acid (5-ALA), catalyzed by ALA synthase (ALAS)4,31. This is the rate limiting step in heme biosynthesis in nonerythroid cells. 5-ALA is then exported out to the cytosol where the next four steps occur to form coproporphyrinogen III (CPgenIII), which is then imported back to the mitochondria, where it is converted into protoporphyrin IX (PPIX). Finally, one molecule of iron is incorporated to the protoporphyrin IX (PPIX) to produce heme, a reaction catalyzed by ferrochelatase (FECH)2,4.
The level of heme biosynthesis depends primarily on the level of ALAS enzyme which is tightly controlled by intracellular iron and heme4. The biosynthesis of heme can be affected by genetic defects, availability of certain minerals and vitamins (e.g., riboflavin, zinc), exposure to toxins (e.g., aluminum, lead), anoxia, fever, and levels of certain steroids (e.g., estrogen)32-35. The level of heme synthesis is altered in various diseased conditions. Decreased heme biosynthesis can cause anemia as well as neurological diseases3,36. Alternatively, increased heme biosynthesis plays an important role in the progression of certain cancers28,37. Heme has been shown to be critical for the growth, differentiation and survival of mammalian adipose, erythroid and neuronal cells4,38-41. For example, heme deficiency leads to neurite damage in primary mouse cortical neurons via the inhibition of glutamate NMDA (N-methyl-D-aspartate) receptor17. Additionally, inhibition of heme synthesis causes programmed cell death in the human epithelial cervix carcinoma HeLa cells26,41. Therefore, measuring the heme biosynthesis levels in various cells under different conditions is important for studying etiology and progression of many diseases.
Here we describe a fast and sensitive method to measure the level of intracellular heme synthesis by using [4-14C] 5-aminolevulic acid. This is an alternative method to other methods using 55Fe or 59Fe. We prefer using 14C because its radiation is very weak. In contrast, strong protection is required for working with Fe isotopes. Furthermore, this method is intended to measure and compare heme synthesis in different cells in parallel in a quick manner. In order to measure absolute heme levels, one may use the previously established method involving the use of HPLC42,43.

<table><tbody><tr><td>Acetone</td><td>Sigma</td><td>650501</td><td></td></tr><tr><td>Diethy ether</td><td>Sigma</td><td>296082</td><td></td></tr><tr><td>HCl (Hydrochloric acid)</td><td>Fisher</td><td>A481-212</td><td></td></tr><tr><td>Liquid Scintillation cocktail&amp;nbsp;</td><td>MP Biomedicals</td><td>882470</td><td></td></tr><tr><td>Trypan blue</td><td>Gibco</td><td>15250</td><td></td></tr><tr><td>Radiolabeled 4-&lt;sup&gt;14&lt;/sup&gt;C aminolevulinic acid</td><td>Perkin-Elmer life sciences</td><td></td><td>Store at -80 &amp;deg;C</td></tr><tr><td>CelLytic M</td><td>Sigma</td><td>C2978</td><td>Mammalian cell lysis reagent</td></tr><tr><td>Pierce BCA Protein Assay Kit&amp;nbsp;</td><td>Thermo Scientific</td><td>23227</td><td></td></tr><tr><td>&lt;strong&gt;Specific reagent&lt;/strong&gt;</td><td>&lt;strong&gt;Component&lt;/strong&gt;</td><td>&lt;strong&gt;Dispense&lt;/strong&gt;</td><td></td></tr><tr><td>Heme extraction buffer:&lt;br /&gt;Acetone:HCl:Water (25:1.3:5)</td><td>Acetone&lt;br /&gt; Concentrated HCl&lt;br /&gt;Water</td><td>25 ml&lt;br /&gt;1.3 ml&lt;br /&gt;5 ml</td><td></td></tr></tbody></table>

measurement of heme synthesis levels in mammalian cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

This method was used to compare the levels of heme synthesis in normal (HBEC30KT) vs. cancer (HCC4017) lung cells. Figure 2 shows a higher level of heme synthesis in cancer cells (HCC4017) than normal lung cells (HBEC30KT). The level of heme synthesis was also measured in normal and cancer cells in the presence of mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Cells were treated with 10 μM CCCP for 24 hr before the measurement of heme synthesis levels. As expected, the levels o...

Watch this Scientific Journal Video about Measurement of Heme Synthesis Levels in Mammalian Cells at JoVE.com

Measurement of Heme Synthesis Levels in Mammalian Cells

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

measurement-of-heme-synthesis-levels-in-mammalian-cells

Research

JoVE Journal

Biology

12.0K Views.  University of Texas at Dallas. Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Video: Measurement of Heme Synthesis Levels in Mammalian Cells

Synthesis and Catalytic Performance of Gold Intercalated in the Walls of Mesoporous Silica

As a promising catalytically active nano reactor, gold nanoparticles intercalated in mesoporous silica (GMS) were successfully synthesized and properties of the materials were investigated. We used a one pot sol-gel approach to intercalate gold nano particles in the walls of mesoporous silica. To start with the synthesis, P123 was used as template to form micelles. Then TESPTS was used as a surface modification agent to intercalate gold nano particles. Following this process, TEOS was added in as a silica source which underwent a polymerization process in acid environment. After hydrothermal processing and calcination, the final product was acquired. Several techniques were utilized to characterize the porosity, morphology and structure of the gold intercalated mesoporous silica. The results showed a stable structure of mesoporous silica after gold intercalation. Through the oxidation of benzyl alcohol as a benchmark reaction, the GMS materials showed high selectivity and recyclability.

Here, we present a protocol with a sol-gel process to synthesize gold intercalated in the walls of mesoporous materials (GMS), which is confirmed to possess a mesoporous matrix with gold intercalated in the walls imparting great stability and recyclability.

As a promising catalytically active nano reactor, gold nanoparticles intercalated in mesoporous silica (GMS) were successfully synthesized and properties ...

Chemistry

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

Medicine

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

Biochemistry

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Immunology and Infection

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS platform more accessible to new users and have expanded the range of applications. For lysate based CFPS systems, cell extracts can be generated from a variety of organisms, harnessing the unique biochemistry of that host to augment protein synthesis. Within the last 20 years, Escherichia coli (E. coli) has become one of the most widely used organisms for supporting CFPS due to its affordability and versatility. Despite numerous key advances, the workflow for E. coli cell extract preparation has remained a key bottleneck for new users to implement CFPS for their applications. The extract preparation workflow is time-intensive and requires technical expertise to achieve reproducible results. To overcome these barriers, we previously reported the development of a 24 hour cell-free autoinduction (CFAI) workflow that reduces user input and technical expertise required. The CFAI workflow minimizes the labor and technical skill required to generate cell extracts while also increasing the total quantities of cell extracts obtained. Here we describe that workflow in a step-by-step manner to improve access and support the broad implementation of E. coli based CFPS.

This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained.

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous ...

Bioengineering

Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The use of experimental animal models of iron overload or deficiency has been instrumental to advance knowledge of the mechanisms involved in the systemic and cellular regulation of iron homeostasis. The measurement of total iron levels in animal tissues is commonly performed with atomic absorption spectroscopy or with a colorimetric assay based on the reaction of non-heme iron with a bathophenanthroline reagent. For many years, the colorimetric assay has been used for the measurement of the non-heme iron content in a wide range of animal tissues. Unlike atomic absorption spectroscopy, it excludes the contribution of heme iron derived from hemoglobin contained in red blood cells. Moreover, it does not require sophisticated analytical skills or highly expensive equipment, and can thus be easily implemented in most laboratories. Finally, the colorimetric assay can be either cuvette-based or adapted to a microplate format, allowing higher sample throughput. The present work provides a well-established protocol that is suited for the detection of alterations in tissue iron levels in a variety of experimental animal models of iron overload or iron deficiency.

Here, a protocol for the measurement of the non-heme iron content in animal tissues is provided, using a simple, well-established colorimetric assay that can be easily implemented in most laboratories.

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The ...

Mouse Brain Histone Modification Quantification Using Flow Cytometry

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

Author Spotlight: Enhancements in Gene Expression Regulation Research

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...

Neuroscience

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.

This protocol outlines how a three-dimensional cell culture system can be used to model, treat, and analyze chromatin modifications in a near-physiological state.

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid ...

Measurement of Heme Synthesis Levels in Mammalian Cells

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