Name: estimating the yield of compounds on the tlc plate via the blue-led illumination technique
Uploaded: 2022-10-06T13:10:00
Description: Watch this Scientific Journal Video about Estimating the Yield of Compounds on the TLC Plate via the Blue-LED Illumination Technique at JoVE.com

This study was supported by the Ministry of Science and Technology, Taiwan (MOST 108-2320-B-110-007-MY3).

The present protocol is described using lovastatin as an example.&#160;Lovastatin was extracted from one-week-old Aspergillus terreus.
1. Compound extraction
NOTE: For details on compound extraction, please see Figure 1.
<ol>
	<li>Culture Aspergillus terreus on the potato dextrose agar (PDA, see Table of Materials) medium at 30 &#176;C.</li>
	<li>Dry the culture at 40 &#176;C for 24 ...

<ol>
	<li>Pyka, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+progress+of+selected+drugs+in+TLC.">Detection progress of selected drugs in TLC.</a> BioMed Research International. 2014, 732078 (2014).</li><li>Hess, A. V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digitally+enhanced+thin-layer+chromatography:+An+inexpensive,+new+technique+for+qualitative+and+quantitative+analysis.">Digitally enhanced thin-layer chromatography: An inexpensive, new technique for qualitative and quantitative analysis.</a> Journal of Chemical Education. 84 (5), 842-847 (2007).</li><li>Ullah, Q., Mohammad, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamins+determination+by+TLC/HPTLC-a+mini-review.">Vitamins determination by TLC/HPTLC-a mini-review.</a> Journal of Planar Chromatography - Modern TLC. 33 (5), 429-437 (2020).</li><li>Chen, Z., Tao, H., Liao, L., Zhang, Z., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quick+identification+of+xanthine+oxidase+inhibitor+and+antioxidant+from+Erycibe+obtusifolia+by+a+drug+discovery+platform+composed+of+multiple+mass+spectrometric+platforms+and+thin-layer+chromatography+bioautography.">Quick identification of xanthine oxidase inhibitor and antioxidant from Erycibe obtusifolia by a drug discovery platform composed of multiple mass spectrometric platforms and thin-layer chromatography bioautography.</a> Journal of Separation Science. 37 (16), 2253-2259 (2014).</li><li>Duncan, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chiral+separations:+A+comparison+of+HPLC+and+TLC.">Chiral separations: A comparison of HPLC and TLC.</a> Journal of Liquid Chromatography. 13 (14), 2737-2755 (1990).</li><li>Sherma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin-layer+chromatography+in+food+and+agricultural+analysis.">Thin-layer chromatography in food and agricultural analysis.</a> Journal of Chromatography A. 880 (1-2), 129-147 (2000).</li><li>Boche&#324;ska, P., Pyka, A., Boche&#324;ska, P., Boche&#324;ska, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+acetylsalicylic+acid+in+pharmaceutical+drugs+by+TLC+with+densitometric+detection+in+UV.">Determination of acetylsalicylic acid in pharmaceutical drugs by TLC with densitometric detection in UV.</a> Journal of Liquid Chromatography. 35 (10), 1346-1363 (2012).</li><li>Poole, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+chromatography+at+the+turn+of+the+century.">Planar chromatography at the turn of the century.</a> Journal of Chromatography A. 856 (1-2), 399-427 (1999).</li><li>Rapid Johnson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=simple+quantitation+in+thin-layer+chromatography+using+a+flatbed+scanner.">simple quantitation in thin-layer chromatography using a flatbed scanner.</a> Journal of Chemical Education. 77 (3), 368-372 (2000).</li><li>El-Gindy, A., Ashour, A., Abdel-Fattah, L., Shabana, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+derivative+spectrophotometric,+TLC-densitometric,+and+HPLC+determination+of+acebutolol+HCL+in+presence+of+its+acid-induced+degradation+product.">First derivative spectrophotometric, TLC-densitometric, and HPLC determination of acebutolol HCL in presence of its acid-induced degradation product.</a> Journal of Pharmaceutical and Biomedical Analysis. 24 (4), 527-534 (2001).</li><li>Elkady, E. F., Mahrouse, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversed-phase+ion-pair+HPLC+and+TLC-densitometric+methods+for+the+simultaneous+determination+of+ciprofloxacin+hydrochloride+and+metronidazole+in+tablets.">Reversed-phase ion-pair HPLC and TLC-densitometric methods for the simultaneous determination of ciprofloxacin hydrochloride and metronidazole in tablets.</a> Chromatographia. 73 (3-4), 297-305 (2011).</li><li>Musharraf, S. G., Ul Arfeen, ., Shoaib, Q., M, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+TLC-densitometric+method+for+the+quantification+of+a+steroidal+drug,+danazol+in+its+pharmaceutical+formulations.">Development and validation of TLC-densitometric method for the quantification of a steroidal drug, danazol in its pharmaceutical formulations.</a> Journal of Planar Chromatography - Modern TLC. 25 (4), 331-337 (2012).</li></ol>

The present study described a new approach, the blue-LED illuminator, to quantify compounds without using expensive and specialized equipment, such as HPTLC, HPLC, and GC method, and the method was compared with the bioassay and UV-detected methods to evaluate quantification performance. As a result, it was concluded that the blue-LED illumination method is a relatively safe and effective protocol used to quantify the yield of targeted compounds on the TLC plate.
Previous studies have reported...

Thin-layer chromatography (TLC) is widely used as a qualitative and quantitative technique in the field of organic chemistry1,2,3. The main advantages of TLC are that it provides fast detection, flexible sample requirements, and does not require specialized equipment4. To date, even though many advanced approaches have been established, TLC is still the main method for identifying unknown samples in a mixture. However, the challenge of this approach is the lack of safe and inexpensive equipment for quantifying the sample yield, especially for developing laboratories with limited budgets. The present study, therefore, aimed to develop an efficient, safe, and inexpensive method combining with TLC to estimate the yield of the samples.
Unlike high-performance TLC (HPTLC), high-performance liquid chromatography (HPLC), and gas chromatography (GC) with strict sample requirements, time-consuming, and involvement of multistep for sample preparation1,5, TLC showed several advantages. First, for sample preparation, the HPLC and GC cannot detect the crude extract because the crude extract may plug the column of HPLC and GC. Second, when the samples are not UV-suitable (important for HPLC analysis) or with low volatility (important for GC analysis), TLC can be applied to these samples, and the use of visualization reagent makes the isolated samples visible on thin layers6,7,8. Third, for general users, HPLC and GC generally require a relatively long time pre-training before working independently, compared to TLC. In addition, quantitative TLC analysis, known as high-performance TLC (HPTLC), can digitize the information on a TLC plate with a highly sensitive scanner. However, the cost of the HPTLC system is relatively expensive. As such, developing a cost-effective and fast approach to quantify samples on the TLC plate is an important topic.
Similar methods have been developed for TLC yield quantification; for example, Johnson9 reported a technique that allows the quantification of the samples on a TLC plate by using a flatbed scanner attached to a computer. In 2001, El-Gindy et al.10 developed the TLC- densitometric method, which was used to detect the compound with optical density, and the technique was also applied by Elkady et al.11. In 2007, Hess2 presented the digitally enhanced-TLC (DE-TLC) method applied to detect the yield of a compound on a TLC plate using a digital camera combined with UV light. Hess also compared the cost differences between HPTLC and DE-TLC method and concluded that the DE-TLC method could be used in high school and college labs because of its affordable cost2. However, the cost of the TLC-densitometric method was still expensive, and the operation of ultraviolet light requires adequate pre-training in case the users might get exposed to ultraviolet radiation. Therefore, compatible with TLC, developing an efficient, safe, and inexpensive method to quantify the sample yield is desirable.
The present study described a protocol for detecting the sample on a TLC plate using the blue-LED illuminator, and developed a regression model with high reliability (high R-square value) to measure the dimensions of the bands, and then determine the compound yield. Finally, it was found that the blue-LED illumination method is a relatively safe (vs. UV-detection method), cheap (vs. GC, HPLC, and HPTLC), and effective (vs. bioassay method) approach for yield quantification.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>American bacteriological Agar</td>
			<td>Condalab</td>
			<td>1802.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspergillus terreus&#160;</td>
			<td></td>
			<td>ATCC 20542</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue-LED illuminator</td>
			<td>MICROTEK</td>
			<td>Bio-1000F</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific&#160;</td>
			<td>HERAEUS Megafuge 8</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact UV lamp</td>
			<td>UVP</td>
			<td>UVGL-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate</td>
			<td>MACRON</td>
			<td>MA-H078-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Paper 125mm</td>
			<td>ADVANTEC</td>
			<td>60311102</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td>Freeware</td>
			<td>https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr>
			<td>Lovastatin standard</td>
			<td>ACROS</td>
			<td>A0404262</td>
			<td></td>
		</tr>
		<tr>
			<td>MiBio Fluo&#160;</td>
			<td>MICROTEK</td>
			<td>V1.04</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Hexane</td>
			<td>C-ECHO</td>
			<td>HH3102-000000-72EC</td>
			<td></td>
		</tr>
		<tr>
			<td>OriginPro</td>
			<td>OriginLab</td>
			<td>9.1</td>
			<td>https://www.originlab.com/origin</td>
		</tr>
		<tr>
			<td>Potato dextrose broth H</td>
			<td>STBIO MEDIA</td>
			<td>110533</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>EYELA</td>
			<td>SB-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Fluka</td>
			<td>30743-2.5L-GL</td>
			<td></td>
		</tr>
		<tr>
			<td>TLC silica gel 60 F254</td>
			<td>MERCK</td>
			<td>1.05554.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Alfa Aesar</td>
			<td>10229873</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic vibration machine</td>
			<td>DELTA</td>
			<td>DC600</td>
			<td></td>
		</tr>
	</tbody>
</table>

estimating the yield of compounds on the tlc plate via the blue-led illumination technique

Thin-layer chromatography (TLC) is an accessible analytical technique that has been extensively used in organic chemistry research to quantify the yield of unknown samples. The present study developed an effective, cheap, and safe method to estimate the yield of samples on a TLC plate using the blue-LED illuminator. Lovastatin extracted from Aspergillus terreus was the example compound used in the present study. Regression models based on the lovastatin standard were used to evaluate the yield of lovastatin. Three methods were compared: bioassay, UV detection, and blue-LED illumination. The result showed that the blue-LED illumination method is significantly more time-effective than UV detection and bioassay methods. Additionally, the blue-LED illumination was a relatively safe option because of the concern of biological hazards in the bioassay method (e.g., microbial infection) and ultraviolet exposure in the UV detection method. Compared to the expensive methods requiring specialized instruments and long-term training before working independently, such as GC, HPLC, and HPTLC, using the blue-LED illuminator was an economical option to estimate the yield of samples from a TLC plate.

This study presented the blue-LED illumination method to estimate the yield of compounds, and this method was validated and compared to bioassay and UV-detected methods (Table 1). The regression models were developed based on the dimensions of bands and concentration of standards for three methods, respectively, to predict the yield of samples. First, in the results of the bioassay method, the R-square between the dimensions of the inhibition zone and lovastatin standards was 0.99, and the sample yield w...

Watch this Scientific Journal Video about Estimating the Yield of Compounds on the TLC Plate via the Blue-LED Illumination Technique at JoVE.com

Estimating the Yield of Compounds on the TLC Plate via the Blue-LED Illumination Technique

The present protocol developed a method to estimate the yield of compounds on the TLC plate using the blue-LED illumination technique. The advantages of this approach are that it is safe, effective, inexpensive, and allows the researcher to measure multiple samples simultaneously.

Estimating the Yield of Compounds on the TLC Plate via the Blue-LED Illumination Technique

Thin-layer chromatography (TLC) is an accessible analytical technique that has been extensively used in organic chemistry research to quantify the yield ...

This protocol developed a method to estimate the yield of compounds on the TLC plate using the blue-LED illumination technique. The advantages of this method are that the blue-LED illumination technique is a safe, effective and cheap method, and allows the researcher to measure multiple samples at the same time. This method also can be applied to biochemistry and natural products chemistry.To begin, dry the culture at 40 degrees Celsius for 24 hours. Transfer the dried culture into a 50 milliliter tube using sterilized tweezers and add 15 milliliters of ethyl acetate. Shake the mixture vigorously by vortexing for one minute and incubate the tube for one hour at 40 degrees Celsius with shaking at 200 RPM.Then sonicate the mixture using a 40 kilohertz ultrasonic bath at 40 degrees Celsius for one hour. Next, centrifuge the mixture at 5, 000 G for one minute at room temperature and filter through an 11 micrometer filter paper. Extract the filtrate with an equal volume of sterile water in a separatory funnel.After phase separation, collect the organic layer, evaporate it in a rotary evaporator, and dissolve the residue in two milliliters of ethyl acetate. Pack the column with normal phase silica gel as the stationary phase, and use n-hexane ethyl acetate and trifluoroacetic acid as the mobile phase. Load two milliliters of the extract onto the column and add the mobile phase solvent at a flow rate of one milliliter per minute to elute the extract.After verifying the effluent by TLC to confirm the presence of lovastatin, evaporate in a rotary evaporator at 45 degrees Celsius until the solvent gets removed. Dissolve the residue in one milliliter of ethyl acetate and then mix with an equal volume of 1%trifluoroacetic acid. Centrifuge the mixture at 5, 000 G for one minute at room temperature, and collect the organic layer in a new glass tube.Using a capillary pipette spot five microliters of samples and lovastatin standards onto the baseline of the TLC plate, leaving a border of one centimeter on the sides of the TLC plate. Then dry the TLC plate in a fume hood for five minutes at room temperature. Place the plate gently by forceps in a saturated glass chamber containing the mobile phase solvent.Cover the chamber with a glass lid and allow the plate to develop fully. Remove the plate from the chamber when the solvents line reaches one centimeter from the top of the plate. Mark the solvent line with a pencil and dry the plate in the fume hood for 10 minutes at room temperature.After drying, immediately soak the plate in 10%sulfuric acid solvent, and then dry in the fume hood for 10 minutes at room temperature. Next, place the plate on the heating panel until the brown spots appear. Transfer the plate to the blue-LED illuminator and scan using compatible freeware.The results obtained from the bioassay method demonstrated that the R square between the dimensions of the inhibition zone and lovastatin standards was 0.99, and the sample yield was 0.56 milligrams as predicted by the regression model. The UV detection method shows that the R square between the lovastatin standards and the dimension of bands on the TLC plate was 0.97, and the sample yield predicted by the regression model was 0.53 milligrams. However, the band's edges were blurry and relatively low signal intensity bands were observed.For the blue-LED illumination method, the R square between lovastatin standards and the band's dimension on the TLC plate was 0.98, and the sample yield was 0.54 milligrams as predicted by the regression model. The predicted yield using the blue-LED illuminator was closer to the bioassay method and clear bands were obtained. If the plate is overheated, the visualization of lovastatin may be difficult to observe.

estimating-yield-compounds-on-tlc-plate-via-blue-led-illumination

Research

JoVE Journal

Biology

Retrograde Labeling of Retinal Ganglion Cells by Application of Fluoro-Gold on the Surface of Superior Colliculus

Retinal ganglion cell (RGC) counting is essential to evaluate retinal degeneration especially in glaucoma. Reliable RGC labeling is fundamental for evaluating the effects of any treatment. In rat, about 98% of RGCs is known to project to the contralateral superior colliculus (SC) (Forrester and Peters, 1967). Applying fluoro-gold (FG) on the surface of SC can label almost all the RGCs, so that we can focus on this most vulnerable retinal neuron in glaucoma. FG is taken up by the axon terminals of retinal ganglion cells and bilaterally transported retrogradely to its somas in the retina. Compare with retrograde labeling of RGC by putting FG at stump of transected optic nerve for 2 days, the interference of RGC survival is minimized. Compare with cresyl violet staining that stains RGCs, amacrine cells and endothelium of the blood vessel in the retinal ganglion cell layer, this labeling method is more specific to the RGC. This video describes the method of retrograde labeling of RGC by applying FG on the surface of SC. The surgical procedures include drilling the skull; aspirating the cortex to expose the SC and applying gelatin sponge over entire dorsal surface of SC are shown. Useful tips for avoiding massive intracranial bleeding and aspiration of the SC have been given.

This video describes the method of retrograde labeling of RGC by applying fluoro-gold (FG) on the surface of superior colliculus (SC). Technique involves drilling the skull, aspirating the cortex, and applying gelatin sponge over entire dorsal surface of SC.

Retinal ganglion cell (RGC) counting is essential to evaluate retinal degeneration especially in glaucoma. Reliable RGC labeling is fundamental for ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.