Name: proteomic analysis of human macrophage polarization under a low oxygen environment
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment at JoVE.com

AM is funded by the Young Group Leader Program (ATIP/Avenir Inserm-CNRS), by la Ligue Nationale contre le Cancer and la Fondation ARC pour la recherche sur le Cancer. We thank Mariette Matondo from the Mass Spectrometry for Biology platform (UTECHS MSBIO, Pasteur Institute, Paris). We thank Lauren Anderson for her reading of the manuscript.

Human blood samples (LRSC) from healthy, de-identified donors were obtained from EFS (French National Blood Service) as part of an authorized protocol (CODECOH DC-2018&#8211;3114). Donors gave signed consent for the use of blood.
1. Media and Buffer Preparation
<ol>
	<li>Prepare the macrophage medium [RPMI glutamax + 10 mM HEPES + 1x non-essential amino acids (NEAA)] and warm it to 37 &#176;C.</li>
	<li>Prepare the macrophage medium + 10% human serum from AB plasma (SAB), filter it (0.22 &#1...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+Polarization.">Macrophage Polarization.</a> Annual Review of Physiology. 79, 541-566 (2017).</li><li>Chinetti-Gbaguidi, G., Staels, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+polarization+in+metabolic+disorders:+functions+and+regulation.">Macrophage polarization in metabolic disorders: functions and regulation.</a> Current Opinion in Lipidology. 22 (5), 365-372 (2011).</li><li>Chow, A., Brown, B. D., Merad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+mononuclear+phagocyte+system+in+the+molecular+age.">Studying the mononuclear phagocyte system in the molecular age.</a> Nature Reviews Immunology. 11 (11), 788-798 (2011).</li><li>Mosser, D. M., Edwards, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+full+spectrum+of+macrophage+activation.">Exploring the full spectrum of macrophage activation.</a> Nature Reviews Immunology. 8 (12), 958-969 (2008).</li><li>Murray, P. J., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+and+pathogenic+functions+of+macrophage+subsets.">Protective and pathogenic functions of macrophage subsets.</a> Nature Reviews Immunology. 11 (11), 723-737 (2011).</li><li>Ginhoux, F., Schultze, J. L., Murray, P. J., Ochando, J., Biswas, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+multidimensional+concept+of+macrophage+ontogeny,+activation+and+function.">New insights into the multidimensional concept of macrophage ontogeny, activation and function.</a> Nature Immunology. 17 (1), 34-40 (2016).</li><li>Xue, 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=Transcriptome-based+network+analysis+reveals+a+spectrum+model+of+human+macrophage+activation.">Transcriptome-based network analysis reveals a spectrum model of human macrophage activation.</a> Immunity. 40 (2), 274-288 (2014).</li><li>Csete, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+in+the+cultivation+of+stem+cells.">Oxygen in the cultivation of stem cells.</a> Annals of the New York Academy of Sciences. 1049, 1-8 (2005).</li><li>Johansson, 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=Functional,+morphological,+and+phenotypical+differences+between+rat+alveolar+and+interstitial+macrophages.">Functional, morphological, and phenotypical differences between rat alveolar and interstitial macrophages.</a> American Journal of Respiratory Cell and Molecular Biology. 16 (5), 582-588 (1997).</li><li>Pfau, J. C., Schneider, J. C., Archer, A. J., Sentissi, J., Leyva, F. J., Cramton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+oxygen+tension+affects+phenotype+in+cultured+bone+marrow-derived+macrophages.">Environmental oxygen tension affects phenotype in cultured bone marrow-derived macrophages.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 286 (2), L354-L362 (2004).</li><li>Grodzki, A. C. G., Giulivi, C., Lein, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+tension+modulates+differentiation+and+primary+macrophage+functions+in+the+human+monocytic+THP-1+cell+line.">Oxygen tension modulates differentiation and primary macrophage functions in the human monocytic THP-1 cell line.</a> PLoS One. 8 (1), e54926 (2013).</li><li>Court, M., Petre, G., Atifi, M. E., Millet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+Signature+Reveals+Modulation+of+Human+Macrophage+Polarization+and+Functions+Under+Differing+Environmental+Oxygen+Conditions.">Proteomic Signature Reveals Modulation of Human Macrophage Polarization and Functions Under Differing Environmental Oxygen Conditions.</a> Molecular &amp; Cellular Proteomics. 16 (12), 2153-2168 (2017).</li><li>Cox, 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=Accurate+proteome-wide+label-free+quantification+by+delayed+normalization+and+maximal+peptide+ratio+extraction,+termed+MaxLFQ.">Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ.</a> Molecular &amp; Cellular Proteomics. 13 (9), 2513-2526 (2014).</li><li>Cox, J., Neuhauser, N., Michalski, A., Scheltema, R. A., Olsen, J. V., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Andromeda:+a+peptide+search+engine+integrated+into+the+MaxQuant+environment.">Andromeda: a peptide search engine integrated into the MaxQuant environment.</a> Journal of Proteome Research. 10 (4), 1794-1805 (2011).</li><li>Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-gel+digestion+for+mass+spectrometric+characterization+of+proteins+and+proteomes.">In-gel digestion for mass spectrometric characterization of proteins and proteomes.</a> Nature Protocols. 1 (6), 2856-2860 (2006).</li><li>Court, M., 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=Toward+a+standardized+urine+proteome+analysis+methodology.">Toward a standardized urine proteome analysis methodology.</a> Proteomics. 11 (6), 1160-1171 (2011).</li><li>Wi&#347;niewski, J. R., Zougman, A., Nagaraj, N., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+sample+preparation+method+for+proteome+analysis.">Universal sample preparation method for proteome analysis.</a> Nature Methods. 6 (5), 359-362 (2009).</li><li>Liebler, D. C., Ham, A. -. J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spin+filter-based+sample+preparation+for+shotgun+proteomics.">Spin filter-based sample preparation for shotgun proteomics.</a> Nature Methods. 6 (11), 785-786 (2009).</li><li>Hubner, N. C., Ren, S., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+separation+with+immobilized+pI+strips+is+an+attractive+alternative+to+in-gel+protein+digestion+for+proteome+analysis.">Peptide separation with immobilized pI strips is an attractive alternative to in-gel protein digestion for proteome analysis.</a> Proteomics. 8 (23-24), 4862-4872 (2008).</li><li>Park, 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=Informed-Proteomics:+open-source+software+package+for+top-down+proteomics.">Informed-Proteomics: open-source software package for top-down proteomics.</a> Nature Methods. 14 (9), 909-914 (2017).</li><li>Richter, A., Sanford, K. K., Evans, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+oxygen+and+culture+media+on+plating+efficiency+of+some+mammalian+tissue+cells.">Influence of oxygen and culture media on plating efficiency of some mammalian tissue cells.</a> Journal of the National Cancer Institute. 49 (6), 1705-1712 (1972).</li><li>Packer, L., Fuehr, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+oxygen+concentration+extends+the+lifespan+of+cultured+human+diploid+cells.">Low oxygen concentration extends the lifespan of cultured human diploid cells.</a> Nature. 267 (5610), 423-425 (1977).</li><li>Lengner, C. 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Because proteomics is a powerful tool to study the expression of different proteins from a whole cell or subcellular compartments, optimization of the cell lysis protocol and digestion of proteins has been addressed by a number of studies. There are three main classes of methods, which include in-gel digestion (digestion of proteins in polyacrylamide gel matrix)17, digestion in solution18 and filter-aided sample preparation19. This last method, at fi...

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis, including removal of apoptotic cells and remodelling of the extracellular matrix1. These cells are characterized by a strong phenotypic plasticity2 that translates into a many possible activation states, which are also called polarizations. The precise molecular description of these various polarizations is a priority in the field of macrophage biology3. It has been proposed to classify these polarizations using the so-called M1/M2 dichotomy, in which M1 represents pro-inflammatory and M2 represents anti-inflammatory macrophages. This model fits well in various pathological situations like acute infections, allergy, and obesity4. However, in chronically inflamed tissues and cancer, it has been demonstrated that this classification is unable to grasp the broad phenotypic repertoire that macrophages present in certain cellular environments5,6,7. The current consensus is that macrophage polarization is better described using a multidimensional model to integrate specific microenvironmental signals8. This conclusion has been confirmed through transcriptomic analysis of human macrophages showing that the M1/M2 model is inefficient in describing the obtained polarizations9.
The study presented aims to provide a protocol to obtain proteomic signatures of various polarizations in human macrophages. We describe how to differentiate human macrophages in environments of various oxygen levels and obtain peptides from the whole macrophage proteome to perform a label-free quantification. This quantification allows the comparison of expression levels of various proteins. As research on stem cells has revealed the importance of oxygen as an environmental key parameter10, we seek to understand how this tissue parameter can influence macrophage polarization in humans. The partial pressure of oxygen has been found to range from 3 to 20% (of total atmospheric pressure) in the human body, where 20% corresponds roughly to what is commonly used in a cell culture incubator (the exact value is around 18.6% while taking the presence of water into account).
Previous work has shown that alveolar differ from interstitial macrophages from functional and morphological point of views11 and that these differences are probably partially due to the different oxygen levels to which they are exposed12. Furthermore, bone marrow-derived macrophages show an increased ability to phagocytize bacteria when exposed to a low oxygen environment12. The opposite effect has been found for THP1-differentiated human macrophages13, but these results support the idea that oxygen is a regulator of macrophage biology and that it is necessary to clarify this role at the molecular level in human macrophages. In a previous study, we have applied a proteomics approach to address these issues. By measuring expression levels for thousands of proteins simultaneously, we highlighted the impact of oxygen on polarization and provided a list of new molecular markers. We were also able to relate these findings to some macrophages functions. Notably, we found that the rate of phagocytosis of apoptotic cells was increased in IL4/IL13-polarized macrophages, which was linked to the upregulation of ALOX15 as revealed by the proteomic analysis14. In the present study, we describe how to perform such an analysis.

<table>
	<tbody>
		<tr>
			<td>Hypoxia Working Station</td>
			<td>Oxford Optronix</td>
			<td>Hypoxylab</td>
			<td></td>
		</tr>
		<tr>
			<td>C6 Flow cytometer</td>
			<td>BD</td>
			<td>Accuri C6</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Agilent Technologies</td>
			<td>5188-6435</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid (FA)</td>
			<td>ARISTAR</td>
			<td>450122M</td>
			<td></td>
		</tr>
		<tr>
			<td>R-250 Coomassie blue</td>
			<td>Biorad</td>
			<td>1,610,436</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharide, E.Coli (LPS)</td>
			<td>Calbiochem</td>
			<td>437627</td>
			<td></td>
		</tr>
		<tr>
			<td>2D clean-up kit</td>
			<td>GE Healthcare</td>
			<td>80-6484-51</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium, glutamax supplement</td>
			<td>Gibco</td>
			<td>61870044</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1 M</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids (NEAA) Solution 100X</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS) 1X</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Harvard Apparatus column Reverse C18 micro spin column</td>
			<td>Harvard Apparatus</td>
			<td>74-4601</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA 0.5 M, pH 8.0</td>
			<td>Invitrogen</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Bis-Tris 4-12%</td>
			<td>Life Technologies SAS</td>
			<td>NP0321 BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>CD14 Microbeads human</td>
			<td>Miltenyi Biotec</td>
			<td>130-050-201</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS separation column LS</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrophage colony-stimulating factor (M-CSF)</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-485</td>
			<td></td>
		</tr>
		<tr>
			<td>Interleukin 4 (IL4)</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-917</td>
			<td></td>
		</tr>
		<tr>
			<td>Interleukin 13 (IL13)</td>
			<td>Miltenyi Biotec</td>
			<td>130-112-410</td>
			<td></td>
		</tr>
		<tr>
			<td>Interferon gamma (INF&#947;)</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-482</td>
			<td></td>
		</tr>
		<tr>
			<td>CD14-FITC (clone T&#220;K4)</td>
			<td>Miltenyi Biotec</td>
			<td>130-080-701</td>
			<td></td>
		</tr>
		<tr>
			<td>MACSmix Tube Rotator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-753</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic Acid (TFA)</td>
			<td>Pierce</td>
			<td>28904</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/Lys-C Mix</td>
			<td>PROMEGA</td>
			<td>V5073</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Mini, EDTA-free Protease Inhibitor cocktail</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr>
			<td>Density Gradient Solution (Histopaque 1077)</td>
			<td>Sigma Aldrich</td>
			<td>10771-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Accumax</td>
			<td>Sigma Aldrich</td>
			<td>A7089-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum from human male AB plasma (SAB)</td>
			<td>Sigma Aldrich</td>
			<td>H4522-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA) solution 30%</td>
			<td>Sigma Aldrich</td>
			<td>A9576-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trisma-base</td>
			<td>Sigma Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>49767</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma Aldrich</td>
			<td>114405</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS) 20%</td>
			<td>Sigma Aldrich</td>
			<td>5030</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>9830</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma Aldrich</td>
			<td>34888</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma Aldrich</td>
			<td>43819</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma Aldrich</td>
			<td>57670</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiourea</td>
			<td>Sigma Aldrich</td>
			<td>T8656</td>
			<td></td>
		</tr>
		<tr>
			<td>CHAPS</td>
			<td>Sigma Aldrich</td>
			<td>C9426</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA Assay Kit</td>
			<td>ThermoFisher</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL sterile plastic pipette</td>
			<td>VWR</td>
			<td>612-1685</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer C Eppendorf</td>
			<td>VWR</td>
			<td>460-0223</td>
			<td></td>
		</tr>
		<tr>
			<td>Sep-Pak tC18 reverse phase cartridges, 100 mg</td>
			<td>Waters</td>
			<td>WAT036820</td>
			<td></td>
		</tr>
	</tbody>
</table>

proteomic analysis of human macrophage polarization under a low oxygen environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

Starting from peripheral blood mononuclear cells (PBMCs) obtained by differential centrifugation, the protocol permits the obtaining of a population of CD14+ monocytes with an assessed purity of more than 98% by flow cytometry (Figure 1). These monocytes are secondarily differentiated toward various polarizations (Figure 2). When a fractionation on gel is chosen, the migration on SDS-page gels is adapted to obtain the ...

Watch this Scientific Journal Video about Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment at JoVE.com

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

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Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

Split-BioID ÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

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

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

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

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 ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...