Name: improvement of bacillus subtilis spore enumeration and label analysis in flow cytometry
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Description: Watch this Scientific Journal Video about Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry at JoVE.com

This study was financed in part by the Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior-Brasil (CAPES)-Finance Code 001; Governo do Estado do Amazonas with resources from Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado do Amazonas-FAPEAM; Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq).&#160;The authors thank the Program for Technological Development in Tools for Health PDTIS-FIOCRUZ for use of its facilities.

See the Table of Materials for details related to all materials, instruments, and software used in this protocol.
1. Flow cytometry setting
<ol>
	<li>Alignment of optical parameters of flow cytometer coupled to a computer
	<ol>
		<li>Log in to Cytometer software.</li>
		<li>From the software workspace, select Cytometer | Startup and wait a few minutes | Clean mode | Sit fluids. 
		&#8203;NOTE: Air bubbles and obstructio...

<ol>
	<li>McKenney, P. T., Driks, A., Eichenberger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bacillus+subtilis+endospore:+assembly+and+functions+of+the+multilayered+coat.">The Bacillus subtilis endospore: assembly and functions of the multilayered coat.</a> Nature Reviews. Microbiology. 11 (1), 33-44 (2013).</li><li>Cutting, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+probiotics.">Bacillus probiotics.</a> Food Microbiology. 28 (2), 214-220 (2011).</li><li>Ricca, E., Baccigalupi, L., Cangiano, G., De Felice, M., Isticato, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+vaccine+delivery+by+non-recombinant+spores+of+Bacillus+subtilis.">Mucosal vaccine delivery by non-recombinant spores of Bacillus subtilis.</a> Microbial Cell Factories. 13, 115 (2014).</li><li>Falahati-Pour, S. K., Lotfi, A. S., Ahmadian, G., Baghizadeh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalent+immobilization+of+recombinant+organophosphorus+hydrolase+on+spores+of+Bacillus+subtilis.">Covalent immobilization of recombinant organophosphorus hydrolase on spores of Bacillus subtilis.</a> Journal of Applied Microbiology. 118 (4), 976-988 (2015).</li><li>Harrold, Z., Hertel, M., Gorman-Lewis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+Bacillus+subtilis+spore+isolation+and+quantifying+spore+harvest+purity.">Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity.</a> Journal of Microbiological Methods. 87 (3), 325-329 (2011).</li><li>Nicholson, W. L., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporulation,+germination+and+outgrowth.">Sporulation, germination and outgrowth.</a> Molecular biological methods for Bacillus. , (1990).</li><li>Mora-Uribe, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+adherence+of+Clostridium+difficile+spores:+the+integrity+of+the+outermost+layer+affects+adherence+properties+of+spores+of+the+epidemic+strain+R20291+to+components+of+the+intestinal+mucosa.">Characterization of the adherence of Clostridium difficile spores: the integrity of the outermost layer affects adherence properties of spores of the epidemic strain R20291 to components of the intestinal mucosa.</a> Frontiers in Cellular and Infection Microbiology. 6, 99 (2016).</li><li>Paidhungat, M., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+ger+proteins+in+nutrient+and+nonnutrient+triggering+of+spore+germination+in+Bacillus+subtilis.">Role of ger proteins in nutrient and nonnutrient triggering of spore germination in Bacillus subtilis.</a> Journal of Bacteriology. 182 (9), 2513-2519 (2000).</li><li>Schnizlein-Bick, C., Spritzler, J., Wilkening, C., Nicholson, J., O'Gorman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+TruCount+absolute-count+tubes+for+determining+CD4+and+CD8+cell+numbers+in+human+immunodeficiency+virus-positive+adults.+Site+Investigators+and+The+NIAID+DAIDS+New+Technologies+Evaluation+Group.">Evaluation of TruCount absolute-count tubes for determining CD4 and CD8 cell numbers in human immunodeficiency virus-positive adults. Site Investigators and The NIAID DAIDS New Technologies Evaluation Group.</a> Clinical and Diagnostic Laboratory Immunology. 7 (3), 336-343 (2000).</li><li>Godfrey, A., Alsharif, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+enumeration+of+viable+spores+by+flow+cytometry.">Rapid enumeration of viable spores by flow cytometry.</a> US Patent. , (2003).</li><li>Karava, M., Bracharz, F., Kabisch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+and+isolation+of+Bacillus+subtilis+spores+using+cell+sorting+and+automated+gating.">Quantification and isolation of Bacillus subtilis spores using cell sorting and automated gating.</a> PLoS ONE. 14 (7), e021989 (2019).</li><li>Genovese, M., Poulain, E., Doppler, F., Toussaint, R., Boyer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+spore+enumeration+using+flow+cytometry:+A+proof+of+concept+for+probiotic+application.">Bacillus spore enumeration using flow cytometry: A proof of concept for probiotic application.</a> Journal of Microbiological Methods. 190, 106336 (2021).</li><li>Trunet, C., Ngo, H., Coroller, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+permeabilization+and+activity+recovery+of+Bacillus+spores+in+adverse+conditions+for+growth.">Quantifying permeabilization and activity recovery of Bacillus spores in adverse conditions for growth.</a> Food Microbiology. 81, 115-120 (2019).</li><li>Tehri, N., Kumar, N., Raghu, H., Vashishth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+of+bacterial+spore+germination.">Biomarkers of bacterial spore germination.</a> Annals of Microbiology. 68, 513-523 (2018).</li><li>Isticato, R., Ricca, E., Baccigalupi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spore+adsorption+as+a+nonrecombinant+display+system+for+enzymes+and+antigens.">Spore adsorption as a nonrecombinant display system for enzymes and antigens.</a> Journal of Visualized Experiments. 145, e59102 (2019).</li><li>Song, 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=Killed+Bacillus+subtilis+spores+as+a+mucosal+adjuvant+for+an+H5N1+vaccine.">Killed Bacillus subtilis spores as a mucosal adjuvant for an H5N1 vaccine.</a> Vaccine. 30 (22), 3266-3277 (2012).</li></ol>

Traditional methods, such as plate counting of colonies, are not only time-consuming, but also need viable cells and do not allow for quantification of inactivated spores5. The Petroff-Hausser chamber is an alternative methodology, but it requires an experienced microscopist to perform it. Flow cytometry has proven to be a useful alternative for this purpose.
Genovese et al.12 described the use of flow cytometry for the quantification of viable c...

Bacillus subtilis is a rod-shaped, gram-positive bacterium that is able to produce quiescent spores when environmental conditions do not allow cell growth1. Spores are extremely stable cell forms and those of several species, including B. subtilis,&#160;are widely used as probiotics for human and animal use2. Due to its resistance and safety properties, the spore of B. subtilis, which displays heterologous proteins, has been proposed as a mucosal adjuvant, a vaccine delivery system, and an enzyme-immobilization platform3,4.
To obtain spores from B. subtilis, it is necessary to expose it to nutrient deprivation using a special culture medium. After obtaining and purifying these spores, one must quantify them to improve test efficiency5,6. Thus, certain methods are applied to analyze the concentration of the spores obtained. Plate counting and a Petroff-Hausser chamber, also known as a counting chamber, can be used. The latter was originally developed to determine the concentration of blood cells; however, it is possible to use it in the field of microbiology for spore counting7,8. Despite being the standard method used for cell counting, reading is laborious since this method is completely manual and its accuracy depends on the experience of the operator.
Flow cytometry-based (FC) analyses have been previously proposed as fast, reliable, and specific approaches for detecting labeled cells of Bacillus spp. The use of flow cytometry counting beads has guaranteed reproducibility in cell counting in routine examinations (absolute count of CD4 and CD8 T lymphocytes) and in the development of research involving particles capable of being detected and counted using flow cytometry9. Godjafrey and Alsharif suggested the use of counting beads for FC quantification of unlabeled spores10. The use of flow cytometry was described for the monitoring of sporulation in Bacillus spp. via&#160;labeling the spore DNA10,11,12,13. Yet another study used FC to evaluate the amount of fluorescently labeled proteins on the spore surface15.
This study sought to use commercial counting beads to ensure a standard of reproducibility with respect to event counting using flow cytometry. Herein, we suggest the use of counting beads for cell counting in FC to refine spore enumeration and evaluate the coupling efficiency of fluorescently labeled antibodies on the spore surface.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>((1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) (EDC)</td>
			<td>Sigma</td>
			<td>341006</td>
			<td></td>
		</tr>
		<tr>
			<td>(N-hydroxysuccinimide) (NHS)</td>
			<td>Sigma</td>
			<td>130672</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human fluorescent antibody</td>
			<td>BioLegend</td>
			<td>501410</td>
			<td>APC anti-human IL-10</td>
		</tr>
		<tr>
			<td>Anti-mouse fluorescent antibody</td>
			<td>Thermo Scientific</td>
			<td>A32723</td>
			<td>Alexa Fluor Plus 488</td>
		</tr>
		<tr>
			<td>BD FACSCanto II</td>
			<td>&#160;BD</td>
			<td></td>
			<td>Flow cytometer</td>
		</tr>
		<tr>
			<td>BD FACSDiva Cytometer Setup &#38; Tracking Beads Kit (use with BD FACSDiva software v 6.x)</td>
			<td>BD</td>
			<td>642412</td>
			<td>Quality control reagent</td>
		</tr>
		<tr>
			<td>BD FACSDiva Software v. 6.1.3</td>
			<td>BD</td>
			<td>643629</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Centrifuge MegaFuge 8R</td>
			<td>Thermo Scientific</td>
			<td>75007213</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting Beads</td>
			<td>BD</td>
			<td>340334</td>
			<td>TruCount Tubes</td>
		</tr>
		<tr>
			<td>Eclipse 80i</td>
			<td>Nikon</td>
			<td></td>
			<td>Fluorescent Microsope</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Ludwig Biotec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Microtubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene tube</td>
			<td>Falcon</td>
			<td>352008</td>
			<td>5 mL polystyrene tube, 12 x 75 mm, without lid, non-sterile</td>
		</tr>
	</tbody>
</table>

improvement of bacillus subtilis spore enumeration and label analysis in flow cytometry

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing need for the development of methodologies that improve the detection of antigens immobilized on the surface of spores together with their quantification. Flow cytometry-based analyses have been previously proposed as fast, reliable, and specific approaches for detecting labeled cells of B. subtilis. Herein, we propose the use of flow cytometry to evaluate the display efficiency of a fluorescent antibody (FA) on the surface of the spore and quantify the number of spores using counting beads. 
For this, we used ethidium bromide as a DNA marker and an allophycocyanin (APC)-labeled antibody, which was coupled to the spores, as a surface marker. The quantification of spores was performed using counting beads since this technique demonstrates high accuracy in the detection of cells. The labeled spores were analyzed using a flow cytometer, which confirmed the coupling. As a result, it was demonstrated that DNA labeling improved the accuracy of quantification by flow cytometry, for the detection of germinated spores. It was observed that ethidium bromide was not able to label dormant spores; however, this technique provides a more precise determination of the number of spores with fluorescent protein coupled to their surface, thus helping in the development of studies that focus on the use of spores as a biotechnological platform in different applications.

In autoclaved spore (AS) samples, 2 &#215; 103 spores/&#181;l and 1 &#215; 103 spores/&#181;l were detected by using counting beads and the Petroff-Hausser method, respectively (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65141/65141fig01.jpg" /> 
Figure 1: General scheme of quantification of spores. (A) Spores labeled with...

Watch this Scientific Journal Video about Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry at JoVE.com

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

This protocol focuses on the use of flow cytometry and counting beads to quantify bacterial spores labeled with ethidium bromide. The method is also efficient for analyzing the covalent coupling of proteins on the surface of intact spores.

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing ...

Cell counter of Bacillus subtilis spores using traditional methods can be a laborious task, as these methods can be completely manual and their accuracies depending on the operator's experience. This protocol makes the enumeration of dormant and germinating spores possible. In addition, the technique also makes it possible to determine the percentage of coping of fluorescent proteins on its surface.Given the setup step present in this protocol the vision demonstrates with facilities and the technical understanding and the detail of care in this execution. Begin by logging into the cytometer software. In the software workspace, select Cytometer, followed by Fluidic Startup.Then choose Cleaning Modes. And finally, initiate SIT Flush. Take 50 microliters of autoclave spores and incubate them with ethidium bromide at a dilution factor of 0.05%volume by volume for 30 minutes protected from light.Wash the spores three times with PBS by centrifuging at 17, 949 G for 10 minutes, and re-suspending in PBS. Next, add 10 microliters of beads following the manufacturer's recommendations for dilution and analyze the sample using a flow cytometer. Define the gating strategy based on the morpho-metric and fluorescence characteristics of the particles using the negative control as a reference.Then mix the tube gently. Attach it to the flow cytometer probe and click Acquire. To set the laser power, go to the parameters tab in the cytometer window, adjust forward scatter to 375 and side scatter to 275.Then select the threshold tab and set it to 500. Next, analyze the dye-free sample containing only spores to eliminate autofluorescence. In the parameter tab, adjust the filter detector three's voltages to 603 and filter detector five's voltages to 538 to differentiate between negative and positive populations using fluorescence in the controls.Then click Compensation, and set filter detector 5 by 3 setup offset to one. To configure the device for acquisition, select Experiment, choose the experiment layout and set the acquisition to 30, 000 events. After adjusting the parameters, acquire data for the samples that are labeled and contain beads in the flow cytometer.Centrifuge 50 microliters of spores at 17, 949 G for 10 minutes. Next, re-suspend the spores using 25 microliters of 1-ethyl-3-3-dimethylaminopropyl carbodiimide and incubate for 15 minutes. Afterward, add 25 microliters of N-hydroxysulfosuccinimide at a concentration of 50 millimolar to the spore suspension.Incubate the spore suspension at room temperature for 30 minutes. Wash the spores three times with PBS by centrifuging as shown earlier. Add fluorescent protein to the samples and incubate overnight at 15 degrees Celsius.Add 10 milligrams per milliliter of ethidium bromide diluted at 1 to 50, then leave the samples on ice for one hour, protected from light. After washing the spores and defining the gates as shown earlier, change the parameters for filter detector three on the x axis, and filter detector five on the Y axis of the dot plot. The counting beads method detected two times 10 to the third spores per microliter in autoclave spore samples.A ethidium bromide staining of autoclave spore samples showed higher mean fluorescence intensity than the staining of non-autoclaved spores, indicating greater staining of genetic material. The autoclave spore surface, coupled with APC labeled, anti-human interleukin 10 antibody showed greater coupling efficiency compared to the non-autoclaved spores. The presence of spores permeable to ethidium bromide indicated the possible presence of germinated spores in the total population.Based on the flow cytometry analyses, it was observed that the fluorescent antibody showed a higher percentage of coupling with the dormant spores in comparison to the germinated ones. Moreover, as the concentration of fluorescent antibodies increased, an increase in the percentage of coupled spores and mean fluorescence intensity was observed. It is important to perform a thorough homogenization of the current beads to ensure precise readings through flow cytometry and accurate enumeration of spores.Standardizing the concentration of antigens attached to spore, Canadian studies analyze the use of spore as vaccine antibodies.

improvement-bacillus-subtilis-spore-enumeration-label-analysis-flow

Research

JoVE Journal

Biochemistry

Identification of Fatty Acids in Bacillus cereus

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the double bond. Changes in FA composition play a crucial role in the adaptation of bacteria to their environment. These modifications entail a change in the ratio of iso versus anteiso branched FAs, and in the proportion of unsaturated FAs relative to saturated FAs, with double bonds created at specific positions. Precise identification of the FA profile is necessary to understand the adaptation mechanisms of Bacillus species.
Many of the FAs from Bacillus are not commercially available. The strategy proposed herein identifies FAs by combining information on the retention time (by calculation of the equivalent chain length (ECL)) with the mass spectra of three types of FA derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl ester (picolinyl). This method can identify the FAs without the need to purify the unknown FAs.
Comparing chromatographic profiles of FAME prepared from Bacillus cereus with a commercial mixture of standards allows for the identification of straight-chain saturated FAs, the calculation of the ECL, and hypotheses on the identity of the other FAs. FAMEs of branched saturated FAs, iso or anteiso, display a constant negative shift in the ECL, compared to linear saturated FAs with the same number of carbons. FAMEs of unsaturated FAs can be detected by the mass of their molecular ions, and result in a positive shift in the ECL compared to the corresponding saturated FAs.
The branching position of FAs and the double bond position of unsaturated FAs can be identified by the electron ionization mass spectra of picolinyl and DMOX derivatives, respectively. This approach identifies all the unknown saturated branched FAs, unsaturated straight-chain FAs and unsaturated branched FAs from the B. cereus extract.

Identification of Fatty Acids in Bacillus cereus

We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl).

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the ...

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis is an obligate pathogen that causes fatal inhalational anthrax. Bacillus cereus is considered a useful model for studying B. anthracis due to its close evolutionary relationship. The gene cluster ba1554 - ba1558 of B. anthracis is highly conserved with the bc1531- bc1535 cluster in B. cereus, as well as with the bt1364-bt1368 cluster in Bacillus thuringiensis, indicating the critical role of the associated genes in the Bacillus genus. This manuscript describes methods to prepare and characterize a protein product of the first gene (ba1554) from the gene cluster in B. anthracis using a recombinant protein of its ortholog in B. cereus, bc1531.

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

Bacillus anthracis is the obligate pathogen of fatal inhalational anthrax, so studies of its genes and proteins are strictly regulated. An alternative approach is to study orthologous genes. We describe biophysicochemical studies of a B. anthracis ortholog in B. cereus, bc1531, requiring minimal experimental equipment and lacking serious safety concerns.

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

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 loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

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

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

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

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample&#39;s impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

This protocol demonstrates real-time recording of full dose-response relationships for agonist-induced GPCR activation from a single cell layer grown on a single microelectrode using label-free impedance measurements. The new dosing scheme significantly increases throughput without loss in time resolution.

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach ...

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An important first step in any membrane-dependent reaction is binding of protein on the phospholipid membrane. An approach to studying protein interaction with lipid membranes has been developed using fluorescently labeled proteins and flow cytometry. This method allows the study of protein-membrane interactions using live cells and natural or artificial phospholipid vesicles. The advantage of this method is the simplicity and availability of reagents and equipment. In this method, proteins are labeled using fluorescent dyes. However, both self-made and commercially available, fluorescently labeled proteins can be used. After conjugation with a fluorescent dye, the proteins are incubated with a source of the phospholipid membrane (microvesicles or cells), and the samples are analyzed by flow cytometry. The obtained data can be used to calculate the kinetic constants and equilibrium Kd. In addition, it is possible to estimate the approximate number of protein binding sites on the phospholipid membrane using special calibration beads.

Here, we describe a set of methods for characterizing the interaction of proteins with membranes of cells or microvesicles.

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An ...

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 &#197; resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 &#197;, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-&#197; resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

High-resolution cryo-EM maps of macromolecules can be also achieved by using 200 kV TEM microscopes. This protocol shows the best practices for setting accurate optics alignments, data acquisition schemes, and selection of imaging areas that are all essential for the successful collection of high-resolution datasets using a 200 kV TEM.

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ...

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...