This work is supported by the Faculty Research Fund of the Swarthmore College [KC] and the Robert Reynolds and Lucinda Lewis &#39;70 Summer Research Fellowship for BJ.

1. Fabrication of the heat block
<ol>
	<li>Wire the 120 V, 100 W strip heater to the rheostat (see Table of Materials).</li>
	<li>Prepare the 20.3 cm x 15.2 cm x 5 cm (8 in x 5 in x 2 in) block of aluminum by drilling 60 holes in a 6 x 10 grid (see Table of Materials). Ensure that the holes are spaced 2 cm from center to center in both directions. Each should be 1.1 cm in diameter and 4.2 cm deep (Figure 1). 
	NOTE: Perform the drilling on a milling machine or drill press with high-speed steel drill bits. The heating element and cooling element were both chosen to cover as much of the contact surface of the 15.2 cm x 5 cm surfaces as possible.</li>
	<li>Drill two additional holes on one of the 20.3 cm x 5 cm surfaces between the 1st and 2nd column and the 9th and 10th column, matching the size of the temperature controller probes (see Table of Materials).</li>
	<li>Construct a case from 1.2 cm (0.5 in) clear acrylic sheets (see Table of Materials) to both hold the elements in place and insulate the completed heat block. Use two layers of acrylic to insulate the back side of the heating element (Figure 1).</li>
	<li>In the final assembly, apply thermal paste (see Table of Materials) to maximize heat conductance from the heating element into the block and from the block to the cooling element.</li>
</ol>
2. Determination of thermal gradient settings
<ol>
	<li>Connect the water bath/aquarium chiller with Tygon tubing (see Table of Materials). Insulate the tubing with foam pipe insulation as needed.</li>
	<li>Insert the thermostat probe into the holes on the side of the aluminum block. Ensure that probe 1 is positioned near the heating element.</li>
	<li>Place microcentrifuge tubes filled to the brim (1.5 mL) with tap water in all the milled holes (60 tubes total).</li>
	<li>Turn on the temperature controller and set the stop heating temperature of probe 1 to 35-37 &#176;C and probe 2 to 21.5-22.5 &#176;C. 
	NOTE: The proposed thermostat has two outlets that operate independently; only probe 1 is used for regulating warm temperature in this particular use case. Therefore, set the temperature of probe 2 to that of the low-end temperature.</li>
	<li>Rotate the rheostat to turn on the heating element and set it to medium.</li>
	<li>Turn on the water bath/aquarium chiller and set the chiller temperature to 15 &#176;C.</li>
	<li>Check that the block is warm on one end and cool on the other after 10 min. 
	CAUTION: The exposed ends of the heating element can be hot; do not touch them.</li>
	<li>Check the temperature inside each microcentrifuge tube using a thermocouple with a K-type electrode (see Table of Materials) every 10 min afterward. The temperature will stabilize after ~60 min and appear linear (Figure 2).</li>
	<li>Adjust the values of the endpoints by changing the settings of the temperature controller and the water bath as needed.</li>
</ol>
3. Thermal exposure and live:dead enumeration
NOTE: Step 2 can be omitted once the desired settings for the temperature gradient are determined.
<ol>
	<li>Turn on the recirculating water bath and heater and set them to 15 &#176;C and 37 &#176;C, respectively, to generate a temperature gradient from 19.5 &#176;C to 37 &#176;C.</li>
	<li>To ensure the thermal gradient is linear, place microcentrifuge tubes filled to the brim (1.5 mL) with tap water in all the milled holes (60 tubes total).</li>
	<li>Let the heat block reach the set temperature by waiting 45-60 min. Check the temperature inside each microcentrifuge tube using a thermocouple with a K-type electrode to see if it has reached the expected temperature. Note these temperatures.</li>
	<li>If the study organisms are &#62;500 &#181;m in size and can be easily transferred from one container to another (e.g., a copepod), fill a 1.5 mL microcentrifuge tube with 750 &#181;L of 0.45 &#181;m filtered seawater. Alternatively, if the study organisms are small, fill a 1.5 mL microcentrifuge tube with 250 &#181;L of 0.45 &#181;m filtered seawater. 
	NOTE: For the representative data, larvae of the sand dollar Dendraster excentrics, which are 2 , 4, and 6 days post-fertilization, were used (see Table of Materials). The average (&#177; S.D., n = 15 for each age) size of these individuals was 152 &#177; 7 &#181;m, 260 &#177; 17 &#181;m, and 292 &#177; 14 &#181;m, respectively. Given these larvae can be easily concentrated (step 3.5), the microcentrifuge tubes were filled with 750 &#181;L of filtered seawater.</li>
	<li>Concentrate the study organisms&#39; culture with reverse filtering (i.e., place the mesh in the container holding the study organisms and remove water through the top of the mesh), so that the study organisms remain in the bottom of the beaker11. 
	NOTE: A 30 &#181;m nylon mesh was used for the larval sand dollars studied (see Table of Materials).</li>
	<li>Rinse the concentrated animal sample with filtered seawater (e.g., when culturing with algal food items or other chemicals). Repeat the reverse filtering once more to concentrate the animal sample.</li>
	<li>Place a known number of individual organisms into the half-filled microcentrifuge tubes. Count the small planktonic organisms under a dissecting microscope (see Table of Materials) and transfer them with glass Pasteur pipettes. 
	NOTE: The number of organisms to place is size dependent; for larval sand dollars that were ~200 &#181;m in size, 20 individuals per microcentrifuge tube was appropriate. 
	CAUTION: Glass pipettes are more desirable than plastic pipettes as some planktonic organisms are hydrophobic and will stick to plastic surfaces.</li>
	<li>Add 0.45 &#181;m filtered seawater to the microcentrifuge tubes containing animals until the final volume is 1 mL.</li>
	<li>To allow the organisms to gradually warm up to the desired experimental temperature, place the microcentrifuge tubes with animals, prepared in step 3.7, into the heat block starting from the cold end. Place pairs of microcentrifuge tubes on each row (12 tubes total).</li>
	<li>Wait 10 min.</li>
	<li>Move the pairs of microcentrifuge tubes inserted at step 3.9 to the adjacent drilled holes with warmer temperatures. Place additional pairs of microcentrifuge tubes in each row at the cold end. Each row will now have four tubes. Wait another 10 min.</li>
	<li>Continue to add microcentrifuge tubes with animals by shifting their positions from the colder end to the warmer end in pairs. Wait 10 min between each shift until the heat block is completely filled. 
	NOTE: Steps 3.9-3.12 are considered a ramping-up phase to increase the temperature experienced by the study organisms gradually.</li>
	<li>Let the animals incubate at the designated temperature for 2 h. This step is the constant temperature exposure phase of the experiment.
	<ol>
		<li>Check the temperature of the microcentrifuge tubes with a thermocouple every hour if the incubation period exceeds 2 h. 
		NOTE: Adjust the incubation time based on the experimental needs. If the incubation is longer than 2 h, check the temperature of the tubes at regular time intervals with a thermocouple in case of unforeseen equipment failure. To minimize disturbance to the study organisms, randomly place six or more microcentrifuge tubes filled only with filtered seawater into the block for temperature monitoring.</li>
	</ol>
	</li>
	<li>At the end of the incubation period, measure the temperature inside each microcentrifuge tube using a thermocouple with a K-type electrode. Note these temperatures.</li>
	<li>Remove all 60 microcentrifuge tubes with animals and place them in pre-labeled holders.</li>
	<li>Incubate the tubes (step 3.14) at the predetermined temperature, such as the rearing temperature, for 1 h, which is the recovery period. 
	NOTE: The recovery period can be species-specific. For the larval sand dollar, the rearing temperature was 18 &#176;C, and thus the sample was placed in an environmental chamber. Consult relevant literature and/or conduct a trial experiment to ensure the live:dead count was not affected by the length of the recovery period. In the representative data, the number of animals alive after 1 h was the same as after 12 or 24 h of recovery.</li>
	<li>To enumerate the proportion of study organism that is alive after the thermal exposure, transfer the contents of an individual microcentrifuge tube onto a 35 mm Petri dish using a glass pipette.</li>
	<li>Observe and note the relative number of individuals that are still active (alive) and those that have seized swimming or dissolved (dead) under a dissecting microscope. Ensure that the total number of individuals observed equals the number of individuals placed into the tubes in step 3.7. Check the side of the microcentrifuge tubes and Petri dish for individuals if the numbers do not match.</li>
</ol>
4. Computation of LT50
<ol>
	<li>Generate a data table in CSV format with at least the following headers: grouping variable of interest, temperature of the tube in &#176;C, number of individuals alive, and number of individuals dead. 
	NOTE: For the representative data, the grouping variable of interest is replaced by age since the goal is to compare between age groups.</li>
	<li>To fit the data with logistic regression, use a generalized linear model with a binomial distribution. Supplementary Coding File 1 shows an example sample script using the open-source software R22.</li>
	<li>To determine the median upper thermal limit (LT50), compute the predictor value (i.e., temperature) at which 50% of the individuals survived. Supplementary Coding File 2 shows an example script using the function dose.p from the MASS23 in R22.</li>
</ol>

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The authors have no conflict of interest to declare.

This protocol provides an accessible and customizable approach to determine the thermal limits of small plankton organisms through acute thermal exposure. The 10-hole design and flexible temperature endpoints, controlled by the water bath at the lower end and the heater at the upper end, enable one to determine LT50 with precision. Using this approach, a difference in the thermal limit that is &lt;1 &#176;C could be detected (Figure 3). This approach provides a rapid determination...

Thermal tolerance is key to organisms&#39; survival and function1,2. As the planet continues to warm due to anthropogenic carbon emissions, increasing attention is being paid to the determination and application of thermal limits3. Various endpoints, such as mortality, failure to develop, and loss of mobility, have been used to determine both upper and lower thermal limits4. These thermal limits are often considered a proxy for an organism&#39;s thermal niche. This information is in turn used to identify species that are more vulnerable to global warming, as well as predict future species distribution and the resulting species interactions3,5,6,7. However, determining thermal limits, especially for small planktonic organisms, can be challenging.
For planktonic organisms, particularly the larval stages of marine invertebrates, the thermal limit can be determined through chronic exposure. Chronic exposure is achieved by rearing larvae at several temperatures over days to weeks and determining the temperature at which larval survivorship and/or developmental rate reduces8,9,10. However, this approach is rather time-consuming and requires large incubators and experience in larval husbandry (see reference11 for a good introduction to culturing marine invertebrate larvae).
Alternatively, acute exposure to thermal stress can be used to determine thermal limits. Often, this determination approach involves placing small vials with larvae in temperature-controlled dry baths12,13,14, leveraging thermal gradient functions in PCR thermal cyclers15,16, or putting glass vials/microcentrifuge tubes along a thermal gradient generated by applied heating and cooling on the ends of large aluminum blocks with holes in which the vials fit snuggly17,18,19. Typical dry baths generate a single temperature; hence, multiple units must be operated simultaneously to assess performance across a range of temperatures. Thermal cyclers generate a gradient but only accommodate a small sample volume (120 &#181;L) and require careful manipulations. Similar to thermal cyclers, large aluminum blocks create linear and stable temperature gradients. Both approaches can be coupled with logistic or probit regression to compute the lethal temperature for 50% percent of the population (LT50)12,20,21. However, the aluminum blocks used were ~100 cm long; this size demands a large lab space and access to specialized computer numerical control milling machines to drill the holes. Together with using two research-grade water baths to maintain the target temperature, the financial cost of assembling the setup is high.
Therefore, this work aims to develop an alternative means to generate a stable, linear temperature gradient with commercially available parts. Such a product must have a small footprint and should be able to be easily used for acute thermal stress exposure experiments for planktonic organisms. This protocol is developed with zooplankton that is &#60;1 mm in size as target organisms, and thus, it was optimized for the use of a 1.5 or 2 mL microcentrifuge tube. Larger study organisms will require containers larger than the 1.5 mL microcentrifuge tubes used and enlarged holes in the aluminum blocks.
In addition to making the experimental apparatus more accessible, this work&#160;aims&#160;to simplify the data processing pipeline. While commercial statistical software provides routines to compute LT50 using logistic or probit regression, the licensing cost is non-trivial. Therefore, an easy-to-use script that relies on the open-source statistical program R22 would make data analysis more accessible.
This protocol shows how a compact heat block can be fabricated with commercially available parts and be applied to exposing zooplankton (larvae of the sand dollar Dendraster excentricus) to acute heat stress to determine their upper thermal limit.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.45 &#181;m membrane filter</td>
			<td>VWR</td>
			<td>74300-042</td>
			<td></td>
		</tr>
		<tr>
			<td>&#189;&#8221; Acrylic sheet</td>
			<td>McMaster-Carr</td>
			<td>8560K266</td>
			<td>Used to construct a ridged case with sufficient insulation.</td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>VWR</td>
			<td>76290-420</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Channel 7 Thermocouple Types Datalogger</td>
			<td>Omega Engineering</td>
			<td>HH506A</td>
			<td>Can be replaced with any thermometer that will fit inside a microcentrifuge tube</td>
		</tr>
		<tr>
			<td>Automatic pipette&#160;</td>
			<td>Ranin&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bolt- and Clamp-Mount Strip Heater 
			with 430 Stainless Steel Sheath, 120V AC, 1-1/2&#34; Wide, 100W</td>
			<td>McMaster-Carr</td>
			<td>3619K32</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal Sea Bioassay Mix</td>
			<td>Pentair</td>
			<td>CM2B</td>
			<td>Use to make aritifical seawater&#160;</td>
		</tr>
		<tr>
			<td>Denraster excentricus</td>
			<td>M-Rep&#160;</td>
			<td></td>
			<td>Sand dollars from California&#160;</td>
		</tr>
		<tr>
			<td>Dissecting microscope&#160;</td>
			<td>Nikon&#160;</td>
			<td>SMZ645</td>
			<td></td>
		</tr>
		<tr>
			<td>DIYhz Aluminum Water Cooling Block, Liquid Water Cooler Heat Sink System for PC Computer CPU Graphics Radiator Heatsink Endothermic Head Silver(40 mm x 120 mm x 12 mm)</td>
			<td>Amazon</td>
			<td></td>
			<td>Connects to water bath and used to cool one end of the block.</td>
		</tr>
		<tr>
			<td>Easy-to-Machine MIC6 Cast Aluminum Sheet 2&#34; thick 8&#34; x 8&#34;&#160;</td>
			<td>McMaster-Carr</td>
			<td>86825K953</td>
			<td>Machined to 2&#34; x 6&#34; x 8&#34; with 60 equally spaced holes (11 mm dia., 42 mm depth) with two addition holes drilled in one side for thermostat probes.</td>
		</tr>
		<tr>
			<td>Economical Flexible Polyethylene Foam Pipe Insulation</td>
			<td>McMaster-Carr</td>
			<td>4530K121</td>
			<td>Covers the plastic tubing between chiller and block to reduce heat loss. Can be omitted if temperature range is close to room temperature&#160;</td>
		</tr>
		<tr>
			<td>EVERSECU 72w 110-240v Aquarium Water Chiller Warmer/Cooler Temperature Controller for Fish Shrimp Tank Marine Coral Reef Tank Below 20 L/30 L Aquarium Chiller</td>
			<td>Amazon</td>
			<td></td>
			<td>Can be used in place of the lab-grade water bath&#160;</td>
		</tr>
		<tr>
			<td>Example with larval sand dollar&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GENNEL 100 g Silver Silicone Thermal Conductive Compound Grease Paste For GPU CPU IC LED Ovens Cooling</td>
			<td>Amazon</td>
			<td></td>
			<td>Improves the thermal conductance between the block and the heating and cooling elements.</td>
		</tr>
		<tr>
			<td>Inkbird WiFi Reptile Thermostat Temperature Controller with 2 Probes and 2 Outlets, IPT-2CH Reptiles Heat Mat Thermostat (Max 250 W per Outlet)</td>
			<td>Amazon</td>
			<td></td>
			<td>Monitors hot and cold ends. Maintains hot end in range</td>
		</tr>
		<tr>
			<td>Lauda Ecoline Silver Air-Cooled Refrigerated Circulators</td>
			<td>VWR</td>
			<td>89202-386</td>
			<td>Can be replaced with an aquarium chiller&#160;</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes</td>
			<td>VWR</td>
			<td>76019-014</td>
			<td>If larger animals are used, scanilation vials (VWR 66022-004) is a good alternative&#160;</td>
		</tr>
		<tr>
			<td>Nitex mesh filter</td>
			<td>&#160;Self made</td>
			<td></td>
			<td>Used hot glue to attached Nitex mesh to 1/2&#34; PVC tubing&#160;</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>VWR</td>
			<td>14673-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (0.35 M)&#160;</td>
			<td>Millpore-Sigma</td>
			<td>P3911-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>R statistical software.&#160;</td>
			<td>The R Project for Statistical Computing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe needle</td>
			<td>VWR</td>
			<td>89219-346</td>
			<td>Depending on size of target organism gague 14 and 16 can be used</td>
		</tr>
		<tr>
			<td>Tygon Tubing&#160;</td>
			<td>McMaster-Carr</td>
			<td>5233K65</td>
			<td>Adjust to match the chiller and block used&#160;</td>
		</tr>
		<tr>
			<td>Zoo Med Repti Temp Rheostat</td>
			<td>Chewy.com</td>
			<td></td>
			<td>Rated to 150 W and rewired to feed directly into the heating element. Used to control rate of heat output</td>
		</tr>
	</tbody>
</table>

thermal limits determination for zooplankton using a heat block

Thermal limits and breadth have been widely used to predict species distribution. As the global temperature continues to rise, understanding how thermal limit changes with acclimation and how it varies between life stages and populations are vital for determining the vulnerability of species to future warming. Most marine organisms have complex life cycles that include early planktonic stages. While quantifying the thermal limit of these small early developmental stages (tens to hundreds of microns) helps identify developmental bottlenecks, this process can be challenging due to the small size of target organisms, large bench space requirement, and high initial fabrication cost. Here, a setup that is geared toward small volumes (mL to tens of mL) is presented. This setup combines commercially available components to generate a stable and linear thermal gradient. Production specifications of the setup, as well as procedures to introduce and enumerate live versus dead individuals and compute lethal temperature, are also presented.

The goal of this protocol is to determine the upper thermal limit of zooplankton. To do so, a stable and linear thermal gradient is needed. The proposed setup was able to generate a thermal gradient ranging from 14 &#176;C to 40 &#176;C by setting the water bath temperature to 8 &#176;C and the heater to 39 &#176;C (Figure 2A). The temperature gradient can be narrowed and shifted by changing the endpoint values. A thermal gradient with a narrower range (19 &#176;C to 37 &#176;C) was also gen...

Watch this Scientific Journal Video about Thermal Limits Determination for Zooplankton Using a Heat Block at JoVE.com

Thermal Limits Determination for Zooplankton Using a Heat Block

The present protocol illustrates the use of commercially available components to generate a stable and linear thermal gradient. Such gradient can then be used to determine the upper thermal limit of planktonic organisms, particularly invertebrate larvae.

Thermal limits and breadth have been widely used to predict species distribution. As the global temperature continues to rise, understanding how thermal ...

thermal-limits-determination-for-zooplankton-using-a-heat-block

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Biology

1.3K Views.  Swarthmore College. The present protocol illustrates the use of commercially available components to generate a stable and linear thermal gradient. Such gradient can then be used to determine the upper thermal limit of planktonic organisms, particularly invertebrate larvae. 

Video: Thermal Limits Determination for Zooplankton Using a Heat Block

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

Rapid PCR Thermocycling using Microscale Thermal Convection

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed1. Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format.
Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations2-9. Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument.
One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time10,11. Here we describe results of our efforts to probe the full 3-D velocity and temperature distributions in microscale convective thermocyclers12. Unexpectedly, we have discovered a subset of complex flow trajectories that are highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone the rate limiting step of PCR. Extremely rapid DNA amplification times (under 10 min) are achievable in reactors designed to generate these flows.

We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

Targeted Studies Using Serial Block Face and Focused Ion Beam Scan Electron Microscopy

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. For many years electron microscopy (EM) has remained an inherently two-dimensional technique. With the advent of serial scanning electron microscope imaging techniques (volume EM), using either an integrated microtome or focused ion beam to slice then view embedded tissues, the third dimension becomes easily accessible. Serial block face scanning electron microscopy (SBF-SEM) uses an ultramicrotome enclosed in the SEM chamber. It has the capability to handle large specimens (1,000 &#181;m x 1,000 &#181;m) and image large fields of view at small X,Y pixel size, but is limited in the Z dimension by the diamond knife. Focused ion beam SEM (FIB-SEM) is not limited in 3D resolution, (isotropic voxels of &#8804;5 nm are achievable), but the field of view is much more limited. This protocol demonstrates a workflow for combining the two techniques to allow for finding individual regions of interest (ROIs) in a large field and then imaging the subsequent targeted volume at high isotropic voxel resolution. Preparing fixed cells or tissues is more demanding for volume EM techniques due to the extra contrasting needed for efficient signal generation in SEM imaging. Such protocols are time consuming and labor intensive. This protocol also incorporates microwave assisted tissue processing facilitating the penetration of reagents, which reduces the time needed for the processing protocol from days to hours.

Here, we present a protocol for efficiently combining serial block face and focused ion beam scanning electron microscopy to target an area of interest. This allows for efficient searching, in three dimensions, and locating rare events in a large field of view.

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. ...

Using a Thermal Camera to Measure Heat Loss Through Bird Feather Coats

Feathers are essential to insulation, and therefore to the cost of thermoregulation,&#160;in birds. There is robust literature on the energetic cost of thermoregulation in birds across a variety of ecological circumstances. However, few studies characterize the contribution of the feathers alone to thermoregulation. Several previous studies have established methods for measuring the insulation value of animal pelts, but they require destructive sampling methods that are problematic for birds, whose feathers are not distributed evenly across the skin. More information is needed about 1) how the contribution of feathers to thermoregulation varies both across and within species and 2) how feather coats may change over space and time. Reported here is a method for rapidly and directly measuring the thermal performance of feather coats and the skin using dried whole skin specimens, without the need to destroy the skin specimen. This method isolates and measures the thermal gradient across a feather coat in a way that measurements of heat loss and metabolic cost in live birds, which use behavioral and physiological strategies to thermoregulate, cannot. The method employs a thermal camera, which allows the rapid collection of quantitative thermal data to measure heat loss from a stable source through the skin. This protocol can easily be applied to various research questions, is applicable to any avian taxa, and does not require destruction of the skin specimen. Finally, it will further the understanding of the importance of passive thermoregulation in birds by simplifying and accelerating the collection of quantitative data.

This protocol describes the quantification of heat transmission through a flat skinned avian specimen using a thermal camera and hot water bath. The method allows obtaining of quantitative, comparative data about the thermal performance of feather coats across species using dried flat skin specimens.

Feathers are essential to insulation, and therefore to the cost of thermoregulation,&#160;in birds. There is robust literature on the energetic cost of ...

High-Throughput Assays of Critical Thermal Limits in Insects

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are essential for predicting responses to climate change. This work describes two high-throughput protocols for measuring insect thermal limits: one for assessing critical thermal minima (CTmin), and the other for assessing heat knock down time (KDT) in response to a static heat stressor. In the CTmin assay, individuals are placed in an acrylic-jacketed column, subjected to a decreasing temperature ramp, and counted as they fall from their perches using an infrared sensor. In the heat KDT assay, individuals are contained in a 96 well plate, placed in an incubator set to a stressful, hot temperature, and video recorded to determine the time at which they can no longer remain upright and move. These protocols offer advantages over commonly used techniques. Both assays are low cost and can be completed relatively quickly (~2 h). The CTmin assay reduces experimenter error and can measure a large number of individuals at once. The heat KDT protocol generates a video record of each assay and thus removes experimenter bias and the need to continuously monitor individuals in real time.

Thermal limits can predict the environments organisms tolerate, which is valuable information in the face of rapid climate change. Described here are high-throughput protocols to assess critical thermal minima and heat knockdown time in insects. Both protocols maximize the throughput and minimize the cost of the assays.

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are ...