This work was supported by Fondo Nacional de Desarrollo Cient&#237;fico y Tecnol&#243;gico (Fondecyt), Chile, Project 11130263 (to CW), Project CONICYT + NERC + Programa de Colaboraci&#243;n Internacional PCI-PII20150073 (to CW) and U-inicia from the Vicerrector&#237;a de Investigaci&#243;n Universidad de Chile (to CW). 
Thanks to Tatiana Naranjo-Palma for the help in the initial stage of this Project, Dr. Jorge Babul and Diego Quiroga-Roger for helpful discussion, Robert Lesch for revising the manuscript and Direcci&#243;n de extensi&#243;n de la Facultad de Ciencias Qu&#237;micas y Farmac&#233;uticas from Universidad de Chile. Thanks too to Marcelo P&#233;rez for editing the video and Neil Stevens for correcting the text and Errol Watson for providing the voice-over.

1. Preparing and Running the Gel
<ol>
	<li>Prepare the non-denaturing 12% polyacrylamide gel gels using the Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) recipe found in Sambrook and Russell14 but using water instead of SDS.
	<ol>
		<li>To prepare 5 mL of resolving gel, use 1.7 mL of distilled water, 2 mL of acrylamide/bis acrylamide (30/0.8% w/v), 1.3 mL of 1.5 M Tris pH 8.8, 0.05 mL of 10% ammonium persulfate, and 0.002 mL of TEMED.</li>
		<li>To prepare 2 mL of stacking gel use 1.5 mL of water, 0.33 mL of acrylamide/bis acrylamide (30/0.8% w/v), 0.25 mL of 1 M Tris pH 6.8, 0.02 mL of 10% ammonium persulfate, and 0.002 mL of TEMED. The dimensions of the gel are 7.3 cm x 8.2 cm x 0.075 cm (l x w x d). 
		Note: Use a 15 well comb of 0.75 mm thickness for the best sensitivity.</li>
	</ol>
	</li>
	<li>Add the appropriate amounts of 6x loading buffer and DNA sample to a 1.5 mL microcentrifuge tube (e.g. use 1 &#181;L of 6x loading buffer to 5 &#181;L of DNA sample).</li>
	<li>Place the gel in the vertical electrophoresis chamber.
	<ol>
		<li>Fill the chamber with 750 mL of 1x TAE buffer to run 2 gels. To prepare 1 L of 50x TAE, use 442 g of Tris base, 57.1 mL of glacial acetic acid, and 100 mL of 0.5 M EDTA pH 8. Add distilled water to a final volume of 1 L.</li>
		<li>Load the samples in the gel. Run the gel at 100 V for 2 h.</li>
	</ol>
	</li>
	<li>Alternatively, prepare a 1x TAE agarose gel using the recipe found in Sambrook14.
	<ol>
		<li>Add 300 mL of 1x&#160;TAE to the horizontal electrophoresis chamber. Run the gel for 45 min at 100 V.</li>
	</ol>
	</li>
</ol>
2. NBT-SG I Staining (Figure 1).
<ol>
	<li>Acrylamide Gel Staining.
	<ol>
		<li>Prepare the solutions: 0.1% w/v NBT and 1.2x SG I. 
		Note: It is possible to use ethidium bromide, GelRed and SYBR Gold instead of SG I, but SG I had the best performance.</li>
		<li>Pour 16.75 mL of 1.2x SYBR Green I solution into a container of appropriate size. 
		Note: We used the lid of a pipette tip box as a container, and these volumes are calculated to cover the gel using this container; its dimensions are 11.6 cm x 7.6 cm x 2.6 cm (l x w x d).</li>
		<li>Place the gel in the solution. Add 3.25 mL of 0.1% w/v NBT solution to give 0.2 mM NBT and 1x (equivalent to 2.0 &#181;M) SG I final concentrations.</li>
		<li>Seal the container with plastic film, and completely wrap the container with aluminum foil to block any ambient light. 
		Note: The plastic film is used to prevent splashing and to avoid reaction of the aluminum foil with the staining solution.</li>
		<li>Shake for 25 min on an orbital shaker at 60 rpm.</li>
		<li>Remove aluminum and plastic cover. Expose to sunlight for 90 min maximum. Check every 5 min until the band of interest appears.</li>
		<li>To use blue light, prepare a protoboard with 3 Light-Emitting Diodes (LED) and place the LEDs about 5 mm from the gel surface (check the staining continuously, as the time needed will depend on the intensity of the LED light source).</li>
	</ol>
	</li>
	<li>Agarose Gel Staining.
	<ol>
		<li>After preparing and running a 1% agarose gel as described in sections 1.4 (the dimensions are 1 cm x 6.2 cm x 7 cm; l x w x d), follow the same steps as in 2.1 with these modifications:</li>
		<li>Pour 41.9 mL of 1.2x SG I solution into the lid of a pipette tip box; its dimensions are 11.6 cm x 7.6 cm x 2.6 cm (l x w x d). Add 8.1 mL of 0.1% NBT solution.</li>
		<li>Seal the container with plastic film, and then completely wrap the container with aluminum foil to block any ambient light.</li>
		<li>Shake for 1 h on an orbital shaker at 60 rpm.</li>
	</ol>
	</li>
</ol>
3. Data Analysis.
<ol>
	<li>NBT-SG I Calibration Curves.
	<ol>
		<li>Obtain gel images by scanning at 1200 dpi.</li>
		<li>Open software for image analysis. Straighten the images using an image editor. Run an image densitometry.</li>
		<li>Calculate the area under the curve. Prepare a graph of Signal/SignalMax versus DNA concentration.</li>
	</ol>
	</li>
</ol>
4. DNA Recovery.
<ol>
	<li>Prepare a 1% agarose gel in 1x TAE using the recipe found in Sambrook14.</li>
	<li>Add 300 mL of 1x&#160;TAE to the horizontal electrophoresis chamber. Load 4 &#181;L of 100 ng/&#181;L pDJ10015 plasmid and DNA ladder following the scheme given in Figure 2. Run the gel for 1 h at 100 V.</li>
	<li>Mark and weigh two 2 mL microcentrifuge tubes.</li>
	<li>Cut the gel down the middle in order to have two identical halves. Each one should contain the ladder and the sample plasmid to compare the different staining techniques. A schematic representation is in Figure 2.</li>
	<li>Stain one half with NBT-SG I as in step 2.
	<ol>
		<li>Cut out the plasmid band using a clean scalpel. Save the gel piece in one tube and weigh.</li>
	</ol>
	</li>
	<li>Stain the other part of the gel with SG I with 50 mL of 1x SG I solution and slowly shake (60 rpm) the gel for 1 h in darkness.
	<ol>
		<li>Place the gel on a transilluminator and expose for 2 min. Cut out the plasmid band using a clean scalpel.</li>
		<li>Save the gel piece in the other tube and weigh. 
		Note: The protocol can be paused here while keeping the tubes at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Carry out plasmid extraction using a commercial gel extraction kit.</li>
</ol>
5. Plasmid Normalization.
<ol>
	<li>In a 1.5 mL microcentrifuge tube, add 10 &#181;L of extracted plasmid and 60 &#181;L of type I water. Mix and transfer to a 60 &#181;L quartz cuvette or use 2 &#181;L directly in a Nanodrop.</li>
	<li>Measure absorption spectra between 230 to 320 nm. Calculate the sample&#39;s plasmid concentration using the following formula: 
	<img alt="Equation 1" src="/files/ftp_upload/56528/56528eq1v3.jpg" /> 
	where D is the dilution factor applied (in this case it is 70/10), Ax is absorption at x nm, and 50 is the conversion factor to &#181;g/mL for dsDNA.</li>
	<li>Dilute samples to 9.1 ng/&#181;L.</li>
	<li>Transform Escherichia coli (E. coli) competent cells (e.g. XL10 Gold) following the Inoue transformation procedure16 using 5 &#181;L of dilute sample.</li>
	<li>Count the number of colonies on the Petri dishes and note the dilution. Calculate the Colony Forming Units (CFU) with the following formula17: 
	<img alt="Equation 2" src="/files/ftp_upload/56528/56528eq2v2.jpg" /> 
	where D is the dilution factor applied from the initial culture.</li>
	<li>Perform an unpaired t-test.</li>
</ol>

<ol>
	<li>LePecq, J. B., Paoletti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescent+complex+between+ethidium+bromide+and+nucleic+acids:+physical-chemical+characterization.">A fluorescent complex between ethidium bromide and nucleic acids: physical-chemical characterization.</a> J. Mol. Biol. 27 (1), 87-106 (1967).</li><li>Singer, V. L., Lawlor, T. E., Yue, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+SYBR+Green+I+nucleic+acid+gel+stain+mutagenicity+and+ethidium+bromide+mutagenicity+in+the+Salmonella/mammalian+microsome+reverse+mutation+assay+(Ames+test).">Comparison of SYBR Green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay (Ames test).</a> Mutat Res. 439 (1), 37-47 (1999).</li><li>Xin, X., Yue, S., Wells, K. S., Singer, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SYBR+Green-I:+a+new+fluorescent+dye+optimized+for+detection+picogram+amounts+of+DNA+in+gels.">SYBR Green-I: a new fluorescent dye optimized for detection picogram amounts of DNA in gels.</a> Biophys. J. 66 (A150), (1994).</li><li>Flores, N., Valle, F., Bolivar, F., Merino, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+DNA+from+agarose+gels+stained+with+methylene+blue.">Recovery of DNA from agarose gels stained with methylene blue.</a> Biotechniques. 13 (2), 203-205 (1992).</li><li>Yang, Y. I., Jung, D. W., Bai, D. G., Yoo, G. S., Choi, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counterion-dye+staining+method+for+DNA+in+agarose+gels+using+crystal+violet+and+methyl+orange.">Counterion-dye staining method for DNA in agarose gels using crystal violet and methyl orange.</a> Electrophoresis. 22 (5), 855-859 (2001).</li><li>Yang, Y. I., 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=Detection+of+DNA+using+a+visible+dye,+Nile+blue,+in+electrophoresed+gels.">Detection of DNA using a visible dye, Nile blue, in electrophoresed gels.</a> Anal. Biochem. 280 (2), 322-324 (2000).</li><li>Santill&#225;n-Torres, 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=A+novel+stain+for+DNA+in+agarose+gels.">A novel stain for DNA in agarose gels.</a> Trends Genet. 9 (2), 40 (1993).</li><li>Adkins, S., Burmeister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+DNA+in+agarose+gels+as+migrating+colored+bands:+applications+for+preparative+gels+and+educational+demonstrations.">Visualization of DNA in agarose gels as migrating colored bands: applications for preparative gels and educational demonstrations.</a> Anal. Biochem. 240 (1), 17-23 (1996).</li><li>Cong, W. T., 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=A+visible+dye-based+staining+method+for+DNA+in+polyacrylamide+gels+by+ethyl+violet.">A visible dye-based staining method for DNA in polyacrylamide gels by ethyl violet.</a> Anal. Biochem. 402 (1), 99-101 (2010).</li><li>Altman, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrazolium+salts+and+formazans.">Tetrazolium salts and formazans.</a> Prog. Histochem. Cytochem. 9 (3), 1-56 (1976).</li><li>Nineham, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemistry+of+formazans+and+tetrazolium+salts.">The chemistry of formazans and tetrazolium salts.</a> Chem. Rev. 55 (2), 355-483 (1955).</li><li>Crandall, I. E., Zhao, B., Vlahakis, J. Z., Szarek, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+imidazole-,+imidazolium,+and+tetrazolium-containing+compounds+with+DNA.">The interaction of imidazole-, imidazolium, and tetrazolium-containing compounds with DNA.</a> Bioorg. Med. Chem. Lett. 23 (5), 1522-1528 (2013).</li><li>Paredes, A. 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=New+visible+and+selective+DNA+staining+method+in+gels+with+tetrazolium+salts.">New visible and selective DNA staining method in gels with tetrazolium salts.</a> Anal. Biochem. 517, 31-35 (2017).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: a Laboratory Manual. , (2012).</li><li>Hansen, W., Garcia, P. D., Walter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+protein+translocation+across+the+yeast+endoplasmic+reticulum:+ATP-dependent+post-translational+translocation+of+the+prepro-&#945;-factor.">In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent post-translational translocation of the prepro-&#945;-factor.</a> Cell. 45 (3), 397-406 (1986).</li><li>Inoue, H., Nojima, H., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+of+Escherichia+coli+with+plasmids.">High efficiency transformation of Escherichia coli with plasmids.</a> Gene. 96 (1), 23-28 (1990).</li><li>Madigan, M., Martinko, J., Parker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Brock biology of microorganisms. , (2003).</li></ol>

The authors have nothing to disclose.

A sensitive and novel DNA staining method based on the reduction of NBT and the use of SG I was presented in this article. The critical step in this protocol is the incubation time of the gel in the NBT-SG I solution and the concentration of NBT. The staining time for agarose gels is longer than for acrylamide gels because of the greater thickness of agarose gels. This method does not work well in the presence of SDS as NBT precipitates in contact with it. If the gel obtains a purple background, we recommend that another...

With the advances in biochemistry and molecular biology, the studies involving DNA have required better techniques to analyze DNA. The first method for DNA staining was silver staining, which is very sensitive but lacks selectivity and does not allow sample recovery. Later, the development of fluorescent DNA staining allowed the selective quantification of DNA with the possibility of sample recovery. One of the first fluorescent dyes used for DNA quantification was ethidium bromide1, which is mutagenic2. However, now there are improved fluorescent dyes that are safer and more sensitive, such as GelRed and SYBR Green I (SG I)3; but all of these fluorescent dyes require the use of an ultraviolet (UV) transilluminator or fluorimeter.
There are other techniques for visibly staining DNA, such as methylene blue4 and crystal violet5,6,7,8,9, but all of these suffer from reduced sensitivity and selectivity.The tetrazolium salts are organic compounds susceptible to reduction, and when this happens they form an insoluble and colored formazan precipitate10,11. Recently, some bivalent tetrazolium salts have been shown to bind to DNA due to their positive tetrazolium rings12.
In a recent publication13, a new visible technique to stain and quantify DNA in polyacrylamide gels was proposed, using the reduction of a bivalent tetrazolium salt called nitro blue tetrazolium (NBT) and fluorescent dyes like ethidium bromide, GelRed, SYBR Green I and SYBR Gold. This reaction worked in the presence of blue light or sunlight and allowed sample recovery with improved quality compared to the use of SG I with a UV transilluminator.The objective of this paper is to provide a detailed protocol of the staining technique using tetrazolium salts.

<table><tbody><tr><td>Nitro blue tetrazolium</td><td>Gold Biotechnology</td><td>298-83-9</td><td>reagent used in the staining</td></tr><tr><td>SYBR Green I 10000X</td><td>Thermo-Fisher</td><td>S7563</td><td>reagent used in the staining</td></tr><tr><td>Gel extraction kit</td><td>QIAGEN</td><td>28704</td><td>used to extract plasmid from the gel in integrity assay</td></tr><tr><td>DNA ladder</td><td>Thermo-Fisher</td><td>SM 1331</td><td>used to make calibration curves and as reference to cut band in integrity assay</td></tr><tr><td>Agar Agar</td><td>Merck</td><td></td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>sodium chloride</td><td>Merck</td><td>1064045000</td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>yeast extract</td><td>Becton, Dickinson and Company</td><td>212750</td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>peptone</td><td>Merck</td><td>72161000</td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>TEMED</td><td>AMRESCO</td><td>761</td><td>used to make polyacrylamide gels</td></tr><tr><td>ammonium persulfate</td><td>Calbiochem</td><td>2310</td><td>used to make polyacrylamide gels</td></tr><tr><td>acrylamide</td><td>invitrogen</td><td>15512-023</td><td>used to make polyacrylamide gels</td></tr><tr><td>bisacrylamide</td><td>SIGMA</td><td>146072</td><td>used to make polyacrylamide gels</td></tr><tr><td>Tris-base</td><td>Merck</td><td>1083821000</td><td>used to make TAE buffer and non-denaturing polyacrylamide gel tris-HCl buffer</td></tr><tr><td>EDTA</td><td>J.T. Baker</td><td>8993-01</td><td>used to make TAE buffer</td></tr><tr><td>Acetic acid</td><td>Merck</td><td>1,000,632,500</td><td>used to make TAE buffer</td></tr><tr><td>agarose</td><td>Merck</td><td>16802</td><td>used to make TAE buffer</td></tr><tr><td>spectrophotometer</td><td>Tecan</td><td>infinite 200 pro</td><td>used to quantify DNA plasmid</td></tr><tr><td>water purificatiom unit</td><td>Merck</td><td>Elix 100</td><td>use to make water type 1 (ASTM) used in spectrophotometry and DNA dilutions\</td></tr><tr><td>distilled water</td><td></td><td>-</td><td>used in gels, culture medium, stainings and general solutions</td></tr><tr><td>HCl</td><td>J.T. Baker</td><td>9535-03</td><td>used to make non-denaturing polyacrylamide gel tris-HCl buffer</td></tr><tr><td>6X DNA loading dye</td><td>Thermo-Fisher</td><td>R0610</td><td></td></tr><tr><td>5 mm Blue LED</td><td>Jinyuan electronics</td><td>SP-021</td><td>light source used in integrity assay</td></tr><tr><td>protoboard</td><td>KANDH</td><td>KH102</td><td>light source support and circuit&amp;nbsp;</td></tr><tr><td>9 V battery</td><td>Duracell</td><td>-</td><td>used to power the LED&#039;s</td></tr><tr><td>horizontal electrophoresis chamber</td><td>Biorad</td><td>1704486EDU</td><td>used to make and run agarose gel in integrity assay</td></tr><tr><td>vertical electrophoresis kit</td><td>Biorad</td><td>1658002EDU</td><td>used to run polyacrylamide gels in calibration curves</td></tr><tr><td>power supply</td><td>Biorad</td><td>1645050&amp;nbsp;</td><td>used to power the electrophoresis</td></tr></tbody></table>

dna staining method based on formazan precipitation induced by blue light exposure

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the formation of a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in a solution of 1x (equivalent to 2.0 &#181;M) SYBR Green I (SG I) and 0.20 mM nitro blue tetrazolium that produces a purple precipitate of formazan when exposed to sunlight or specifically blue light. Also, DNA recovery tests were performed using an ampicillin resistant plasmid in an agarose gel stained with our method. A larger number of colonies was obtained with our method than with traditional staining using SG I with ultraviolet illumination. The described method is fast, specific, and non-toxic for DNA detection, allowing visualization of biomolecules to the "naked eye" without a transilluminator, and is inexpensive and appropriate for field use. For these reasons, our new DNA staining method has potential benefits to both research and industry.

A purple formazan precipitate appears where the DNA is located (verified by mass spectrometry13). In the experiment, a DNA ladder was loaded to make a calibration curve (Figure 3). The protocol works for both acrylamide and agarose gels, but it has a lower intensity and takes a longer time in agarose. However, the use of agarose gels allows the recovery of the sample using a commercially available kit, and it does not interfere with th...

Watch this Scientific Journal Video about DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure at JoVE.com

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

This method allows selective staining and quantification of DNA in gels by soaking the gel in a SYBR Green I/Nitro Blue Tetrazolium solution and then exposing it to sunlight or a blue light source. This produces a visible precipitate and requires almost no equipment, making it ideal for field use.

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

dna-staining-method-based-on-formazan-precipitation-induced-blue

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Biochemistry

9.4K Views.  Universidad de Chile. This method allows selective staining and quantification of DNA in gels by soaking the gel in a SYBR Green I/Nitro Blue Tetrazolium solution and then exposing it to sunlight or a blue light source. This produces a visible precipitate and requires almost no equipment, making it ideal for field use.

Video: DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining. This article demonstrates how to use thiazole orange to stain DNA. Thiazole orange compares favorably to common staining methods, in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.

Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Immunology and Infection

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Bioengineering

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Analysis of Lipid Droplet Content in Fission and Budding Yeasts using Automated Image Processing

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are used as models in lipid metabolic research or for industrial lipid production. Lipid droplets are highly dynamic storage bodies and their cellular content represents a convenient readout of the lipid metabolic state. Fluorescence microscopy is a method of choice for quantitative analysis of cellular lipid droplets, as it relies on widely available equipment and allows analysis of individual lipid droplets. Furthermore, microscopic image analysis can be automated, greatly increasing overall analysis throughput. Here, we describe an experimental and analytical workflow for automated detection and quantitative description of individual lipid droplets in three different model yeast species: the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus, and the budding yeast Saccharomyces cerevisiae. Lipid droplets are visualized with BODIPY 493/503, and cell-impermeable fluorescent dextran is added to the culture media to help identify cell boundaries. Cells are subjected to 3D epifluorescence microscopy in green and blue channels and the resulting z-stack images are processed automatically by a MATLAB pipeline. The procedure outputs rich quantitative data on cellular lipid droplet content and individual lipid droplet characteristics in a tabular format suitable for downstream analyses in major spreadsheet or statistical packages. We provide example analyses of lipid droplet content under various conditions that affect cellular lipid metabolism.

Here, we present a MATLAB implementation of automated detection and quantitative description of lipid droplets in fluorescence microscopy images of fission and budding yeast cells.

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are ...

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can be generated from FMN photolysis upon irradiation with visible light. The ROS generated from FMN photolysis are harmful to microorganisms, including pathogenic bacteria such as Staphylococcus aureus (S. aureus). This article presents a protocol for deactivating S. aureus, as an example, via photochemical reactions involving FMN under visible light irradiation. The superoxide radical anion (<img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" />) generated during the FMN photolysis is evaluated via nitro blue tetrazolium (NBT) reduction. The microbial viability of S. aureus that is attributed to reactive <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" /> species was used to determine the effectiveness of the process. The bacterial inactivation rate is proportional to FMN concentration. Violet light is more efficient in inactivating S. aureus than blue light irradiation, while the red or green light does not drive FMN photolysis. The present article demonstrates FMN photolysis as a simple and safe method for sanitary processes.

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Here, we present a protocol to inactivate pathogenic bacteria with reactive oxygen species produced during photolysis of flavin mononucleotide (FMN) under blue and violet light irradiation of low intensity. FMN photolysis is demonstrated to be a simple and safe method for sanitary processes.

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

Biology

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...