This work developing this protocol was supported by the National Institutes of Health (R01-EB019453-02), the Office of the Director of National Intelligence, Intelligence Advanced Research Projects Activity through Raytheon BBN Technologies Corp (N66001-18-C-4507), the Chan-Zuckerberg Biohub Investigator Program, Defense Advanced Research Projects Agency through Texas A&#38;M University (W911NF1920013), and Centers for Disease Control and Prevention through Johns Hopkins University Applied Physics Laboratory (75D30-11-9C-06818 (CDC3)). The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the above organizations or the U.S. Government. The U.S. Government is authorized to reproduce and distribute reprints for governmental purposes notwithstanding any copyright annotation therein.

1. Preparation of hydrogel particles for particle templated emulsification.
Hydrogel particles used for particle templated emulsification can be prepared using two different methods.
<ol>
	<li>Preparation using commercially available particles
	<ol>
		<li>Add 0.5 g of dried polyacrylamide particles compatible with PTE (e.g., Bio-Gel P-60 Gel (Bio-Rad), 45-90 &#181;m diameter) to 30 mL of sterile water in a 50 mL conical tube and mix well. Incubate at room temperature for 30 min.</li>
	</ol>
	</li>
	<li>Preparation using microfluidic fabrication of particles 
	NOTE: Polyacrylamide particles compatible with PTE can be prepared using commercially available drop makers (e.g., QX200 Drop Generator (Bio-Rad), RayDrop (Fluigent) etc.), or by custom microfluidic design.
	<ol>
		<li>Fabrication of custom master
		<ol>
			<li>Design a soft photolithography mask using computer-aided design (CAD) software. Print the photomask with a 10 &#181;m resolution on circuit board film.</li>
			<li>Pour 1 mL of photoresist onto the center of a 3 in silicon wafer. Use a spin coater to create a 50 &#181;m layer of photoresist by spinning it at 500 rpm for 30 sec followed by 1250 rpm for 30 sec. Place the wafer onto a hotplate set to 95 &#176;C for 15 min to evaporate the solvent.</li>
			<li>Secure the photomask onto the silicon wafer with a cover glass slide and expose the wafer under a collimated 190 mW, 365 &#181;m UV LED for 2.5 min. Place the wafer on a hotplate set to 95 &#176;C for 5 min for post exposure baking.</li>
			<li>Develop the photoresist-silicon wafer by immersing it in a bath of 100% propylene glycol monomethyl ether acetate (PGMEA) for up to 15 min. Rinse the wafer with fresh 100% PGMEA followed by 100% isopropanol. Air dry the wafer.</li>
			<li>Remove any residual isopropanol by drying the wafer on a hotplate set to 95 &#176;C for 1 min. Place the wafer into a clean 3 in Petri dish.</li>
		</ol>
		</li>
		<li>Fabrication of the custom microfluidic device
		<ol>
			<li>Mix the polydimethylsiloxane (PDMS) silicon base and curing reagent in a 10:1 ratio by mass. Degas the mixed PDMS using a desiccator under house vacuum until no air bubbles are observable.</li>
			<li>Pour the degassed PDMS over the master in the petri dish, ensuring the silicon wafer is completely submerged. Degas the silicon wafer and PDMS to remove any air bubbles that may have formed during pouring.</li>
			<li>Cure the PDMS by placing the silicon wafer and PDMS into an oven set to 65 &#176;C for at least 60 min. Excise a block of PDMS containing the microfluidic features from the petri dish using a scalpel. Take extra care to avoid damaging any features present on the silicon master.</li>
			<li>Punch the inlets and the outlets into the PDMS block corresponding to the inlets and outlets in the microfluidic device using a 0.75 mm biopsy punch. Remove any dust and particulates with the repetitive application and removal of packaging tape to the surface of the PDMS block.</li>
			<li>Clean a 50 mm x 75 mm glass slide by rinsing it with 100% isopropanol and subsequently air drying the surface. Plasma treat both the glass slide and the PDMS (features facing up) using 1 mbar of O2 plasma for 1 min using a plasma bonder.</li>
			<li>Affix the PDMS to the glass slide by placing the plasma treated PDMS with features facing down onto the glass slide, plasma treated side facing up. Place the slide into an oven set to 65 &#176;C for at least 30 min to complete the bonding.</li>
			<li>Treat all microfluidic channels with a fluorinated surface treatment to ensure surface hydrophobicity and prevent wetting. Bake the device at 65 &#176;C for at least 10 min.</li>
		</ol>
		</li>
		<li>Fabrication of templating particles
		<ol>
			<li>Prepare a polyacrylamide (PAA) solution consisting of 6.2% acrylamide, 0.18% N,N&#8242;-methylenebis (acrylamide), and 0.3% ammonium persulfate. Load this solution into a 1 mL syringe with a 28G needle.</li>
			<li>Prepare an insoluble continuous phase consisting of 5% (w/w) fluorosurfactant and 1% N,N,NN-tetramethylethylenediamine (TEMED) in hydrofluoroether (HFE) oil for the generation and stabilization of droplets. Load the solution into new 1 mL syringe.</li>
			<li>Load both PAA and HFE solution containing syringes into syringe pumps (E.g., NE-501). Connect both syringes to the microfluidic device using polyethylene tubing inserted onto the syringe and into the device. Before the connecting, prime the pumps to remove the air from the tubing. 
			NOTE: Depending on the model, syringe pumps may be controlled with built in input, manufacture software, or a custom script (available at https://github.com/AbateLab/Pump-Control-Program).</li>
			<li>Run the drop generation device with PAA and HFE oil inputs at 300 &#181;L/h and 500 &#181;L/h, respectively. Collect 1 mL of the droplets in a 15 mL collection tube and incubate for 3 h at room temperature for polymerization. After the incubation, remove the lower layer of oil by pipetting.</li>
			<li>Add 1 mL of 20% (v/v) perfluoro-1-octanol (PFO) in HFE oil to the 15 mL collection tube as a chemical demulsifier. After mixing, spin down the 15 mL collection tube at 2000 x g for 2 min. Remove the PFO/HFE supernatant by pipetting. Repeat 1x.</li>
			<li>Add 2 mL of 2% sorbitan monooleate in hexane to the 15 mL collection tube and vortex to mix. Spin the tube at 3000 x g for 3 min. Remove the supernatant by pipetting to remove surfactant/hexane solution. Repeat 2x.</li>
			<li>Add 5 mL of TEBST buffer (20 mM Tris-HCl pH 8.0, 274 mM NaCl, 5.4 mM KCl, 20 mM EDTA, 0.2% Triton X-100) and mix well. Spin down at 3,000 x g for 3 min. Remove the supernatant by pipetting. Repeat 3x.</li>
			<li>Resuspend in 5 mL TEBST. This solution may be stored at 4 &#176;C indefinitely.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Particle templated emulsification.
Following the preparation of templating particles, PTE is used to encapsulate the sample and reagents in droplets.
<ol>
	<li>Prepare the polyacrylamide particles for particle templated emulsification by centrifuging at 6000 x g for 1 min to pellet the particles, then remove the supernatant by pipetting and resuspend using sterile water. Repeat 3x to ensure removal of any residual TEBST.</li>
	<li>Determine the concentration and diameter of the templating particles using a hemocytometer (or equivalent). Calculate individual particle diameter by measuring the diameters in pixels and converting to microns. Conversion of pixels to microns may be calculated using the hemocytometer (or equivalent) as a calibration slide and measuring the known grid distance in pixels.</li>
	<li>Prepare the disperse phase in a fresh 1.5 mL microcentrifuge tube using a PCR master mix, the appropriate primers, and a fluorescein hydrolysis probe according to Table 1. Incubate at room temperature for 5 min under gentle agitation (10 rpm) using a tube rotator to ensure homogenous distribution of the components. 
	&#8203;NOTE: The volume and target concentration of particles is based upon Poisson loading. As general rule, the number of particles should be an order of magnitude more than the number of samples to be encapsulated. For samples of unknown concentrations, a dilution series is necessary to ensure Poisson loading.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Volume</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>100 &#956;L</td>
			<td>Particles (450 particles / &#956;L)</td>
		</tr>
		<tr>
			<td>200 &#956;L</td>
			<td>2x PCR master mix</td>
		</tr>
		<tr>
			<td>18 &#956;L</td>
			<td>10 &#956;M forward primer</td>
		</tr>
		<tr>
			<td>18 &#956;L</td>
			<td>10 &#956;M reverse primer</td>
		</tr>
		<tr>
			<td>18 &#956;L</td>
			<td>10 &#956;M probe</td>
		</tr>
		<tr>
			<td>0.8 &#956;L</td>
			<td>Triton X100</td>
		</tr>
		<tr>
			<td>45.2 &#956;L</td>
			<td>Nuclease free water</td>
		</tr>
	</tbody>
</table>
Table 1. Preparation of the PCR master mix used with PTE for digital droplet PCR.
<ol start="4">
	<li>Centrifuge the disperse phase at 6000 x g for 1 min and remove the supernatant. Record the volume of the supernatant extracted and using the total disperse phase volume calculated in 2.3 determine the pellet volume. 
	NOTE: The amount of supernatant extracted will vary depending on particle packing, diameter, and concentration with a minimum expected volume of 300 &#181;L.</li>
	<li>Add 1 &#181;L of 1.62 pg /&#181;L Saccharomyces cerevisiae genomic DNA to the pellet from 2.4 and mix thoroughly by pipetting or vigorous tapping. 
	NOTE: The presence of excess aqueous content can decrease encapsulation efficiency. If the sample volume exceeds 1% of the pellet volume, concentrate the sample. If the sample cannot be concentrated, scale the PCR master mix and resulting pellet volume according to the sample volume. PTE permits the emulsification of small (10 &#181;L) to large (2 mL) volumes of templating particles. The PCR master mix (2.3) and oil (2.6) can be scaled according to the target (2.3) and measured (2.4) volume of the particle pellet respectively.</li>
	<li>Add 200 &#181;L 2% fluorosurfactant in HFE oil to the tube as the insoluble continuous phase for emulsification. Ensure the pellet is dislodged by pipetting or tapping/flicking the tube. Then vortex at 3000 rpm for 30 sec. 
	NOTE: The setting corresponding to 3000 rpm may vary depending on brand and model.</li>
	<li>Allow for the emulsions to settle for 1 min. Remove 100 &#181;L of the bottom oil phase and replace this volume with fresh 2% fluorosurfactant in HFE oil. Gently invert the tube several times to mix. Repeat 3-5x or until small satellite droplets have been removed.</li>
</ol>
3. Digital droplet PCR and analysis.
<ol>
	<li>After 2-5 min of settling, remove the bottom oil phase. Replace this volume with 5% fluorosurfactant in fluorocarbon oil (e.g., FC-40).</li>
	<li>Using a wide bore pipette tip, carefully pipette the 100 &#181;L of sample into 200 &#181;L PCR tubes. Place the PCR tubes into a thermocycler and run according to Table 2.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Temperature</td>
			<td>Duration</td>
			<td>Notes</td>
		</tr>
		<tr>
			<td>1</td>
			<td>95 &#176;C</td>
			<td>2 min</td>
			<td></td>
		</tr>
		<tr>
			<td>2</td>
			<td>95 &#176;C</td>
			<td>30 s</td>
			<td></td>
		</tr>
		<tr>
			<td>3</td>
			<td>50 &#176;C</td>
			<td>90 s</td>
			<td></td>
		</tr>
		<tr>
			<td>4</td>
			<td>72 &#176;C</td>
			<td>60 s</td>
			<td></td>
		</tr>
		<tr>
			<td>5</td>
			<td></td>
			<td></td>
			<td>Repeat x34 steps 2 to 4</td>
		</tr>
		<tr>
			<td>6</td>
			<td>72 &#176;C</td>
			<td>2 min</td>
			<td></td>
		</tr>
		<tr>
			<td>7</td>
			<td>4 &#176;C</td>
			<td>hold</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2. Thermocycling conditions for digital droplet PCR using PTE emulsions.
<ol start="3">
	<li>Pipette the sample onto a counting slide using a wide bore pipette tip for fluorescent imaging. Image the sample using a fluorescent microscope with 490 nm excitation and 525 nm emission detection wavelengths.</li>
	<li>Quantify the positive fluorescent droplets (Np) and total drops (NT) to verify the presence and calculate the number of template molecules (&#955;) using the fraction of positive droplets (Np/NT) and Poisson statistics: 
	<img alt="Equation 1" src="/files/ftp_upload/62248/62248eq01.jpg" /></li>
	<li>Calculate the 95% confidence interval (zc = 1.96) by: 
	<img alt="Equation 2" src="/files/ftp_upload/62248/62248eq02.jpg" /></li>
	<li>Calculate the sample concentration (molecules/&#181;L) using the volume (&#957; in &#181;L) of sample added in step 2.5, using the equation given below. Determine the mean and standard deviation of the sample concentration using technical replicates. 
	<img alt="Equation 3" src="/files/ftp_upload/62248/62248eq03.jpg" /></li>
</ol>

<ol>
	<li>Mashaghi, S., Abbaspourrad, A., Weitz, D. A., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+microfluidics:+A+tool+for+biology,+chemistry,+and+nanotechnology.">Droplet microfluidics: A tool for biology, chemistry, and nanotechnology.</a> Trends in Analytical Chemistry. 82, 118-125 (2016).</li><li>Gielen, F., 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=Ultrahigh-throughput-directed+enzyme+evolution+by+absorbance-activated+droplet+sorting+(AADS).">Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS).</a> Proceedings of the National Academy of Sciences of USA. 113 (47), 7383-7389 (2016).</li><li>Mai, S., Murphy, T. W., Lu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+for+genome-wide+studies+involving+next+generation+sequencing.">Microfluidics for genome-wide studies involving next generation sequencing.</a> Biomicrofluidics. 11 (2), 021501 (2017).</li><li>Olmedillas-L&#243;pez, S., Garc&#237;a-Arranz, M., Garc&#237;a-Olmo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+and+emerging+Applications+of+Droplet+Digital+PCR+in+Oncology.">Current and emerging Applications of Droplet Digital PCR in Oncology.</a> Molecular Diagnosis and Therapy. 21 (5), 493-510 (2017).</li><li>Tong, Y., Shen, S., Jiang, H., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Digital+PCR+in+Detecting+Human+Diseases+Associated+Gene+Mutation.">Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation.</a> Cellular Physiology and Biochemistry. 43 (3), 1718-1730 (2017).</li><li>Yan, Y., 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=Evaluation+of+droplet+digital+PCR+for+non-invasive+prenatal+diagnosis+of+phenylketonuria.">Evaluation of droplet digital PCR for non-invasive prenatal diagnosis of phenylketonuria.</a> Analytical and Bioanalytical Chemistry. 411 (27), 7115-7126 (2019).</li><li>Shang, L., Cheng, Y., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+Droplet+Microfluidics.">Emerging Droplet Microfluidics.</a> Chemical Reviews. 117 (12), 7964-8040 (2017).</li><li>The, S., Lin, R., Hung, L., Lee, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+Microfluidics.">Droplet Microfluidics.</a> Lab Chip. 8 (2), 198-220 (2008).</li><li>Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+PCR+hits+its+stride.">Digital PCR hits its stride.</a> Nature Methods. 9, 541-544 (2012).</li><li>Kolodziejczyk, A. A., Kim, J. K., Svensson, V., Marioni, J. C., Teichmann, S. 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+Technology+and+Biology+of+Single-Cell+RNA+Sequencing.">The Technology and Biology of Single-Cell RNA Sequencing.</a> Molecular Cell. 58 (4), 610-620 (2015).</li><li>Hatori, M. N., Kim, S. C., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle-Templated+Emulsification+for+Microfluidics-Free+Digital+Biology.">Particle-Templated Emulsification for Microfluidics-Free Digital Biology.</a> Analytical Chemistry. 90 (16), 9813-9820 (2018).</li><li>Panda, 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=Stop-flow+lithography+to+generate+cell-laden+microgel+particles.">Stop-flow lithography to generate cell-laden microgel particles.</a> Lab Chip. 8 (7), 1056-1061 (2008).</li><li>Yozwiak, C. E., Hirschhorn, T., Stockwell, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+microparticle-based+system+for+pooled+assays+of+small+molecules+in+cellular+contexts.">Towards a microparticle-based system for pooled assays of small molecules in cellular contexts.</a> ACS Chemical Biology. 13 (3), 761-771 (2018).</li></ol>

Authors have nothing to disclose.

PTE uses particles to encapsulate samples in monodispersed droplets by vortexing. In addition to its simplicity and accessibility, PTE provides several additional benefits, including allowing large volumes of droplets to be generated instantaneously. Moreover, the process can be conducted in an isolated tube, obviating the need to transfer samples to microfluidic devices, streamlining the overall workflow and limiting opportunities for sample contamination or loss. The templating particles also provide a means by which to engineer the contents of the resultant droplet reactions. For example, particle size, chemistry, and wettability can be engineered for targeted biomolecule or cell capture, while functional moieties such as enzymes, actives, or nucleic acids, can be displayed on particle to facilitate reactions, such as for single cell sequencing or functional characterization. While the approach is flexible, there are nevertheless important constraints to its use. For example, it is not currently possible to perform droplet additions as are often conducted with microfluidics, requiring that all reaction components be introduced before encapsulation; this requires that reagents be compatible and stable until the droplets can be generated and, in the case of troublesome combinations, can often be addressed by quickly mixing and emulsifying the sample on ice. Alternatively, reactive components that can be triggered externally with light or heat can be used13. PTE thus provides a flexible and scalable method for conducting droplet assays accessible to non-experts. This, coupled with its innate simplicity and flexibility, makes PTE ideal for the execution and development of numerous droplet applications.

Droplet microfluidics leverages compartmentalization in picoliter droplets to increase the sensitivity and accuracy of assays compared to bulk reactions, and have numerous applications in chemical screening, protein engineering, and next generation sequencing1,2,3. For example, digital droplet polymerase chain reaction (ddPCR) affords increased accuracy compared to bulk quantitative polymerase chain reaction (qPCR), with applications for genetic variation in cancers, detection of disease causing mutations, and prenatal diagnostics4,5,6. A challenge of droplet microfluidics, however, is the requirement of microfluidic devices to partition samples; while microfluidics afford excellent control over droplet properties, they require specialized expertise to build and operate7,8. Consequently, droplet-based methods are largely limited to expert labs or, in rare instances, applications in which a commercial instrument is available9,10. To broaden the use of droplet assays, the requirement for specialized microfluidic instrumentation is a hurdle that must be overcome.
In this article, we describe Particle Templated Emulsification (PTE), a microfluidic-free method for performing reactions in monodispersed droplets. In PTE, templating particles engulf the sample into droplets in carrier oil by simple vortexing (Figure 1). As the system mixes, the aqueous portion fragments into droplets of reducing size until the droplets contain single particles, at which point further fragmentation is not possible because it requires breaking the particles. The engulfed sample surrounds the particles as a shell in the droplets, thereby encapsulating any dispersed cells, reagents, or functional moieties (Figure 1D). Thus, PTE requires no equipment or expertise to perform droplet reactions beyond a common vortexer. Additionally, droplet generation takes seconds compared to minutes or hours with microfluidics, and the amount produced is proportional to the container volume, not device operation time, making it supremely scalable. These benefits make PTE ideal for conducting droplet assays in a variety of circumstances in which microfluidics are impractical. Here, we demonstrate PTE and use it to conduct ddPCR.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62248/62248fig01.jpg" /> 
Figure 1. Overview of particle templated emulsification process. (A) Templating particles are mixed with reagents. (B) Excess reagents are removed following centrifugation. (C) The addition of template molecules occurs before the addition of oil. (D) Vortexing produces droplets containing a single template molecule. (E) Subsequent thermocycling and imaging allows for digital droplet analysis of target template. <a href="https://www.jove.com/files/ftp_upload/62248/62248fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 um syringe filter</td>
			<td>Milipore Sigma</td>
			<td>SLGP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5M EDTA, pH 8.0</td>
			<td>Thermo-Fisher</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>0.75 mm biopsy punch</td>
			<td>World Precision Instruments</td>
			<td>504529</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringes</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>1H,1H,2H-Perfluoro-1-Octanol (PFO)</td>
			<td>Sigma-Aldrich</td>
			<td>370533</td>
			<td></td>
		</tr>
		<tr>
			<td>1M Tris-HCI, pH 8.0</td>
			<td>Thermo-Fisher</td>
			<td>15568025</td>
			<td></td>
		</tr>
		<tr>
			<td>27 gauge needles</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>3&#34; silicon wafers, P type, virgin test grade</td>
			<td>University Wafers</td>
			<td>447</td>
			<td></td>
		</tr>
		<tr>
			<td>3D-printed centrifuge syringe holder</td>
			<td>(custom)</td>
			<td>(custom)</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide solution,40%, for electrophoresis, sterile-filtered</td>
			<td>Sigma-Aldrich</td>
			<td>A4058-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A3678-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquapel (fluorinated surface treatment)</td>
			<td>Pittsburgh Glass Works</td>
			<td>47100</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Sigma-Aldrich</td>
			<td>139386</td>
			<td></td>
		</tr>
		<tr>
			<td>FC-40 fluorinated oil</td>
			<td>Sigma-Aldrich</td>
			<td>F9755</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>109827</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N&#8242;-Methylenebis(acrylamide)</td>
			<td>Sigma-Aldrich</td>
			<td>146072-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Novec-7500 Engineering Fluid (HFE oil)</td>
			<td>3M</td>
			<td colspan="2">98-0212-2928-5</td>
		</tr>
		<tr>
			<td>polyethylene tubing</td>
			<td>Scientific Commodities</td>
			<td>B31695-PE/2</td>
			<td></td>
		</tr>
		<tr>
			<td>fluorosurfactant</td>
			<td>Ran Biotechnologies</td>
			<td colspan="2">008-FluoroSurfactant</td>
		</tr>
		<tr>
			<td>PGMEA developer</td>
			<td>Sigma-Aldrich</td>
			<td>484431</td>
			<td></td>
		</tr>
		<tr>
			<td>Photomasks</td>
			<td>CadArt Servcies</td>
			<td>(custom)</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum Multiplex PCR Master Mix (Taq Master Mix)</td>
			<td>Applied Biosystems</td>
			<td>4464263</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Specialty Coating Systems</td>
			<td>G3P-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Span 80 (sorbitane monooleate)</td>
			<td>Sigma-Aldrich</td>
			<td>s6760</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3025 photoresist</td>
			<td>Kayaku</td>
			<td>17030192</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100 (octylphenol ethoxylate)</td>
			<td>Sigma-Aldrich</td>
			<td>t8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20 (polysorbate 20)</td>
			<td>Sigma-Aldrich</td>
			<td>p2287</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum Multiplex PCR Master Mix (Taq Master Mix)</td>
			<td>Applied Biosystems</td>
			<td>4464263</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast FWD</td>
			<td>IDT</td>
			<td colspan="2">5&#8242;-GCAGACCAGACCAGAACAAA-3&#8242;</td>
		</tr>
		<tr>
			<td>Yeast REV</td>
			<td>IDT</td>
			<td colspan="2">5&#8242;-ACACGTATGTATCTAGCCGAATA 
			AC-3</td>
		</tr>
		<tr>
			<td>Yeast Probe</td>
			<td>IDT</td>
			<td colspan="2">5&#8242;-/56-FAM/ATATGTTGT/ZEN/TCACTCGCGCCTGGG/3IABk 
			FQ/-3&#8242;</td>
		</tr>
		<tr>
			<td>EVOS FL AUTO</td>
			<td>Life Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS LED Cube, GFP</td>
			<td>Life Technologies</td>
			<td>&#160;AMEP4651</td>
			<td></td>
		</tr>
		<tr>
			<td>SYLGARD 184 KIT 1.1 LB (PDMS base and curing reagents)</td>
			<td>&#160;Dow Corning</td>
			<td>DC4019862</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Thermo Fisher</td>
			<td>17919</td>
			<td></td>
		</tr>
		<tr>
			<td>Saccharomyces cerevisiae genomic DNA</td>
			<td>Milipore</td>
			<td>69240-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Expanded plasma cleaner (plasma bonder)</td>
			<td>Harrick Plasma</td>
			<td colspan="2">PDC-002 (230V)</td>
		</tr>
	</tbody>
</table>

particle templated emulsification enables microfluidic-free droplet assays

Reactions performed in monodispersed droplets afford enhanced accuracy and sensitivity compared to equivalent ones performed in bulk. However, the requirement of microfluidics to form controlled droplets imposes a barrier to non-experts, limiting their use. Here, we describe particle templated emulsification, an approach to generate monodisperse droplets without microfluidics. Using templating hydrogel spheres, we encapsulate samples in monodispersed droplets by simple vortexing. We demonstrate the approach by using it to perform microfluidic-free digital PCR.

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62248/62248fig02.jpg" /> 
Figure 2. Encapsulation of sample into droplets using particle templated emulsification. (A) templating particles used for particle templating emulsification. (B) Separation of templating particle pellet from supernatant following centrifugation. (C) Droplets resulting from particle templated emulsification with (D) identifiable aqueous shell. <a href="https://www.jove.com/files/ftp_upload/62248/62248fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In PTE, the monodispersity of the emulsions is dictated by that of the templating particles, because the droplets have a diameter slightly larger than the particles. Thus, uniform particles are central to controlled PTE encapsulation11. A variety of methods exist for generating uniform templating particles, including chemical (sol-gel, emulsion polymerization), hydrodynamic (membrane emulsification, homogenization), and filtration methods. Microfluidic approaches in particular, afford superb monodispersity (Figure 2A) and allow additional particle engineering to enhance their functionality in PTE12. Alternatively, templating particles can be purchased, although their uniformity, while adequate, is typically less than with microfluidic generation11.
To perform PTE, the particles are mixed with the sample to be encapsulated (Figure 1A), and the excess supernatant is removed by centrifugation and pipetting (Figure 1B), as illustrated by a photograph of a particle pellet at the bottom of a PCR tube (Figure 2B). The encapsulating oil containing a stabilizing surfactant is then added (Figure 1C), and the sample gently pipetted before vortexing for 30 seconds (Figure 1D), to generate the emulsion (Figure 2C). The resultant droplets contain a particle core and aqueous shell comprising the initial sample, within which reside the reagents, target molecules, and cells necessary for the reaction (Figure 2D). Just as in droplet microfluidic encapsulation, discrete entities like small beads or cells are encapsulated randomly and in accordance with a Poisson distribution, although nearly all droplets contain a templating particle due to the nature of PTE physics.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62248/62248fig03.jpg" /> 
Figure 3. Identification and cleanup of particle templated emulsification droplets. (A) Example of non-uniform droplet generation with multiple particles per droplet from insufficient vortexing. (B) Expected presence of satellites and droplets following particle templated emulsification and (C) the water-in-oil fractionation. (D) Resulting emulsion following oil washing. (E) Excessive satellite generation resulting from residual supernatant during particle templated emulsification. <a href="https://www.jove.com/files/ftp_upload/62248/62248fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Even in successful PTE, double or triple core droplets exist, though they generally contribute negligibly to the reaction, provided they are rare. Achieving a low frequency of multicore droplets while retaining adequate shells requires optimization of process parameters, including surface tension, inter-particle adhesion forces, sample viscosity, container size, and vortexing power and time. For example, a poorly optimized emulsification may contain polydispersed droplets with many templating particles (Figure 3A), indicating that the vortexing was insufficient to fully emulsify the sample. In such instances, detergents can be added to reduce inter-particle adhesion and lower surface tension, or vortexing power or time can be increased. Another common issue is generation of excessive satellites, which are small empty droplets (Figure 3B). Satellites can be unavoidable in PTE emulsions depending on the interfacial tension and rheological properties of the sample and carrier oil. However, they often result from not adequately removing excess sample prior to emulsification (Figure 2B), or vortexing with too much power, stripping the shells from the droplets. In a successful PTE emulsification, satellites should comprise no more than ~10% of the total encapsulated sample volume (Figure 3C)11. At this level, they usually contribute negligibly to the reaction and can be ignored. For aesthetic purposes, they can be cleared from the emulsion by washing with fresh oil (Figure 3D).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62248/62248fig04.jpg" /> 
Figure 4. Evaluation of particle templated emulsification digital droplet PCR. (A) Fluorescent imaging of the droplets identifies positive fluorescent droplets and negative non-fluorescent droplets. (B) Identification of rare template or low concentrations of template with digital droplet PCR. (C) Over abundant template encapsulation resulting in a variable number of template molecules per droplet. <a href="https://www.jove.com/files/ftp_upload/62248/62248fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To demonstrate the utility of PTE, we used it to perform microfluidic-free digital PCR11. Using the process, we encapsulated a sample comprising S. cerevisiae genomic DNA, and thermocycled it. In digital PCR, droplets containing amplified targets become fluorescent, while those without remain dim. Thus, a fluorescent droplet indicates a target, allowing direct quantitation of targets by counting positive droplets (Figure 4A). The number of fluorescent droplets thus scales with the target molecules, yielding few positives when the target is rare (Figure 4B) and many when it is abundant (Figure 4C). As with encapsulation of other discrete components, target encapsulation follows a Poisson distribution, allowing the positive droplet fraction to be transformed into the target concentration (Figure 4D), thereby demonstrating the ability to perform digital PCR with PTE11.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62248/62248fig5v2.jpg" /> 
Figure 5. Demonstration of digital droplet PCR using commercially available PAA. (A) Fluorescent imaging of the droplets identifies negative non-fluorescent droplets. (B) Identification of low concentrations of template with digital droplet PCR. (C) Identification of high concentrations of template with digital droplet PCR. <a href="https://www.jove.com/files/ftp_upload/62248/62248fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
These results are repeatable using commercially available polyacrylamide particles (Figure 5) and demonstrate the ability of PTE to perform standard digital PCR with commercially available polyacrylamide particles, achieving accurate measurements over the same range.
Supplemental File.&#160; <a href="https://www.jove.com/files/ftp_upload/62248/S1.tif" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Particle Templated Emulsification enables Microfluidic-Free Droplet Assays at JoVE.com

Particle Templated Emulsification enables Microfluidic-Free Droplet Assays

Water-in-oil droplet assays are useful for analytical chemistry, enzyme evolution, and single cell analysis, but typically require microfluidics to form the droplets. Here, we describe particle templated emulsification, a microfluidic-free approach to perform droplet assays.

Reactions performed in monodispersed droplets afford enhanced accuracy and sensitivity compared to equivalent ones performed in bulk. However, the ...

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5.4K Views.  University of California. Water-in-oil droplet assays are useful for analytical chemistry, enzyme evolution, and single cell analysis, but typically require microfluidics to form the droplets. Here, we describe particle templated emulsification, a microfluidic-free approach to perform droplet assays. 

Video: Particle Templated Emulsification enables Microfluidic-Free Droplet Assays

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the  dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies.

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

We have developed a strategy to purify and image a large number of centrioles in different orientations amenable for super-resolution microscopy and single-particle averaging.

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell ...

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying cell composition, discrimination of functional states, and molecular pathways underlying observed tissue functions and animal behaviors. However, the isolation of intact, healthy single cells from adult mammalian tissues for subsequent downstream single cell molecular analysis can be challenging. This protocol describes the general processes and quality control checks necessary to obtain high-quality adult single cell preparations from the nervous system or skin that enabled subsequent unbiased single cell RNA sequencing and analysis. Guidelines for downstream bioinformatic analysis are also provided.

This protocol describes the general processes and quality control checks necessary for preparing healthy adult mammalian single cells for droplet-based, high throughput single cell RNA-Seq preparations. Sequencing parameters, read alignment, and downstream single-cell bioinformatic analysis are also provided.

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying ...

Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral lipids encased by a monolayer of phospholipids and proteins. Hepatic LD lipids, such as ceramides, and proteins are implicated in several diseases that cause hepatic steatosis. Although previous methods have been established for LD isolation, they require a time-consuming preparation of reagents and are not designed for the isolation of multiple subcellular compartments. We sought to establish a new protocol to enable the isolation of LDs, endoplasmic reticulum (ER), and lysosomes from a single mouse liver.
Further, all reagents used in the protocol presented here are commercially available and require minimal reagent preparation without sacrificing LD purity. Here we present data comparing this new protocol to a standard sucrose gradient protocol, demonstrating comparable purity, morphology, and yield. Additionally, we can isolate ER and lysosomes using the same sample, providing detailed insight into the formation and intracellular flux of lipids and their associated proteins.

This protocol establishes a novel method of lipid droplet isolation and purification from mouse livers, using a well-established endoplasmic reticulum isolation kit.

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...

Quantification of Circular RNAs Using Digital Droplet PCR

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 water-in-oil droplets, and the PCR occurs in the individual droplets. The dd-PCR has several advantages over conventional real-time qPCR, including increased accuracy in detecting low-abundance targets, omitting reference genes for quantification, eliminating technical replicates for samples, and showing high resilience to inhibitors in the samples. Recently, dd-PCR has become one of the most popular methods for accurately quantifying target DNA or RNA for gene expression analysis and diagnostics. Circular RNAs (circRNAs) are a large family of recently discovered covalently closed RNA molecules lacking 5' and 3' ends. They have been shown to regulate gene expression by acting as sponges for RNA-binding proteins and microRNAs. Furthermore, circRNAs are secreted into body fluids, and their resistance to exonucleases makes them serve as biomarkers for disease diagnosis. This article aims to show how to perform divergent primer design, RNA extraction, cDNA synthesis, and dd-PCR analysis to accurately quantify specific circular RNA (circRNA) levels in cells. In conclusion, we demonstrate the precise quantification of circRNAs using dd-PCR.

This protocol describes the detailed method of digital droplet PCR (dd-PCR) for precise quantification of circular RNA (circRNA) levels in cells using divergent primers.

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 ...

Label-Retention Expansion Microscopy (LR-ExM) Enables Super-Resolution Imaging and High-Efficiency Labeling

Expansion microscopy (ExM) is a sample preparation technique that can be combined with most light microscopy methods to increase the resolution. After embedding cells or tissues in swellable hydrogel, samples can be physically expanded three-to sixteen-fold (linear dimension) compared to the original size. Therefore, the effective resolution of any microscope is increased by the expansion factor. A major limitation of the previously introduced ExM is reduced fluorescence after polymerization and the digestion procedure. To overcome this limitation, label-retention expansion microscopy (LR-ExM) has been developed, which prevents signal loss and greatly enhances labeling efficiency using a set of novel trifunctional anchors. This technique allows one to achieve higher resolution when investigating cellular or subcellular structures at a nanometric scale with minimal fluorescent signal loss. LR-ExM can be used not only for immunofluorescence labeling, but also with self-labeling protein tags, such as SNAP- and CLIP-tags, thus achieving higher labeling efficiency. This work presents the procedure and troubleshooting for this immunostaining-based approach, as well as discussion of self-labeling tagging approaches of LR-ExM as an alternative.

Label-Retention Expansion Microscopy LR-ExM Enables Super-Resolution Imaging and High-Efficiency Labeling

A protocol of label retention expansion microscopy (LR-ExM) is demonstrated. LR-ExM uses a novel set of trifunctional anchors, which provides better labeling efficiency compared to previously introduced expansion microscopies.

Expansion microscopy (ExM) is a sample preparation technique that can be combined with most light microscopy methods to increase the resolution. After ...