DIY cryogenic retrieval device had been tested in course of the projects "Modeling bacterial protein prenylation in eukaryotic cells using bioinformatics and molecular approaches" sponsored by Mary Louise Andrews Award for Cancer Research program, Virginia Academy of Science, "Physiological Meaning of RNA Editing in Plasma Dendritic Cells" sponsored by The Thomas F. Jeffress and Kate Miller Jeffress Memorial Trust and State Contract 16.740.11.0364 (Ministry of Science and Education, Russia). We would like to thank Beth Eom for assistance with the RNA integrity experiment.

1. Assembly of Cryotolerant Device for Retrieval of Cryoboxes
<ol>
 <li>Using pliers, straighten out one side of 3 sided strong-tie (Figure 1A) to make an L shaped strong-tie shown in Figure 1B. Note: The dimensions of strong-tie can be selected depending on the diameter of the neck of Dewar.</li>
 <li>Secure two such strong-ties with a strong-tie T strap (Figure 2) using Crown bolt nut, washer and screw to form the Cryoscoop base as shown in Figure 3(A-B). Note: Two strong ties are used to allow retrieval of 5 &frac12; in. X 5 &frac12; in. cryoboxes.</li>
 <li>Using a slotted plate (Figure 4), align the slots on the strong-tie T strap with the slots on one end of the slotted plate and secure the two together with help of Hillman nut and bolts (Figure 5). Note: If the slotted plate and strong tie's does not come with pre-drilled holes, you will have to have holes drilled using carbide drill bit.</li>
 <li>Bend the other end of the slotted plate with the help of pliers as shown in Figure 6. (This step is optional).</li>
 <li>Wrap the handle with foam to better grasp the device when in liquid nitrogen as shown in Figure 6. (optional) Note: Remove the foam prior to autoclaving.</li>
</ol>
2. Assembly of Cryotolerant Device for Retrieval of Cryovials
The Cryotolerant DIY retrieval device can also be adapted for retrieval of cryovials. This involves substituting the flat base with a stainless steel strainer as shown altered for scooping out vials from liquid nitrogen (Figure 7).
<ol>
 <li>Unscrew the Crown nuts and bolts attaching the strong-tie T strap to the slotted plate.</li>
 <li>Before attaching the stainless steel strainer to the slotted plate, gently bend the neck of the strainer with the help of pliers to orient the base at an angle to resemble a ladle (Figure 8). Note: If the strainer comes with a lip, use pliers to bend the lip down; this will make maneuvering with the device easier.</li>
 <li>Align the hole on handle of the strainer along with the holes on the slotted plate and secure the strainer with help of Hillman nuts and bolts as shown in Figure 8. Note: if the strainer does not come with pre-drilled holes, you will need to arrange to have holes drilled with carbide drill bit.</li>
</ol>
3. Retrieving Cryoboxes and Cryovials From Dewar
<ol>
 <li>To begin, put on cryogenic gloves and remove 1 or 2 freezer racks from the Dewar to allow room for maneuvering.</li>
 <li>Gently scrape along the bottom of the Dewar with the upright Cryotolerant DIY retrieval device fitted with strong-tie T strap base (Figure 5) and collect the box fallen to the bottom of the Dewar tank. Note: Liquid nitrogen boils immediately on contact with a warmer object, enveloping the object in insulating nitrogen gas (Leidenfrost effect). To cool the device prior to retrieval of samples, slowly immerse the device into the Dewar and allow liquid nitrogen to cool the device sufficiently before proceeding with the above step.</li>
 <li>Pull the device upright along the walls of the Dewar to scoop the freezer box out of the tank.</li>
 <li>Grab the box retrieved from the bottom of the Dewar.</li>
 <li>For retrieving cryovials, gently scrape the bottom of the Dewar with the cryoscoop fitted with a strainer (Figure 9).</li>
 <li>Similar to cryobox retrieval, pull the device upright and grab the vials collected in the strainer.</li>
</ol>
4. Representative Results
A major challenge in gene expression studies is that the quality of RNA depends on the storage conditions for both frozen tissue samples and extracted RNA4. Figure 10 shows the effect of sample handling on integrity of total RNA. Total RNA from the same tissue source (human adipose) was subjected to different sample handling. Prior to experiment, intact total RNA preparate shows clear 28S and 18S rRNA bands with the 28S rRNA band approximately twice as intense as the 18S rRNA band (Figure 10, lane 1). This 2:1 ratio (28S:18S) is an indication that the RNA is intact, while partially degraded RNA appears as a smear, or its 28S:18S intensity ratio departs from 2:15. Lanes 3-4 depicts RNA samples with significant degradation. These RNA samples were subjected to repeated partial defreezing by short-term retrievals from the Dewar (Method A), thus modeling long term storage with periodic decanting due to necessity of the retrieval of the fallen vials and boxes (Figure 10, Lane 3-4). However, samples stored in Dewar serviced using DIY device (Method B) and, thus, no partial defreezing, contain intact RNA (Figure 10, Lane 2).
<img alt="Figure 1" src="/files/ftp_upload/3903/3903fig1.jpg" /> 
Figure 1. A) The base consists of two 3 sided strong-tie of shown dimensions. Each strong-tie contains one large pre-drilled hole (solid arrow) and multiple smaller holes (small arrow) on each face. This provides an option to allow fastening with nut and bolts. Also the smaller holes allow rapid drainage of liquid nitrogen during sample retrieval. B) One side of each of the strong-tie is straightened with the help of pliers to make L shaped strong-tie.
<img alt="Figure 2" src="/files/ftp_upload/3903/3903fig2.jpg" /> 
Figure 2. Strong-tie T strap is used to secure the strong-ties together. Like strong- tie, T-strap has one large pre-drilled hole (solid arrow) and two smaller holes (small arrow) on each corner. This allows securing strong-ties along the length of the T - Strap using nuts and bolt. It also enables fastening T-strap to the slotted plate.
<img alt="Figure 3" src="/files/ftp_upload/3903/3903fig3.jpg" /> 
Figure 3. A) Each strong-tie is fastened to one corner of T-strap, along its length using crown bolt nuts, bolts and washers. B) Two strong-ties are secured adjacent to each other to help retrieve 5 &frac12; in. X 5 &frac12; in. cryoboxes. For smaller cryoboxes such as 2 in. X 2 in. , a single strong-tie can be fastened directly to the slotted plate without a T-strap.
<img alt="Figure 4" src="/files/ftp_upload/3903/3903fig4.jpg" /> 
Figure 4. Slotted flat plate is used as the handle. Length of the slotted plate depends on the depth of the Dewar. In this case, 4 feet of slotted plate was used. The thickness of slotted plate is critical since thin slotted plates (&lt;14 gauges) make the device flimsy while retrieving samples from liquid nitrogen filled Dewar.
<img alt="Figure 5" src="/files/ftp_upload/3903/3903fig5.jpg" /> 
Figure 5. The flat base consisting of strong-ties and T strap is then secured at one end of the slotted plate. The larger hole on the remaining corner of T strap is aligned with the slot on the flat plate and fastened using hex bolt and nut. This device can be now used to retrieve cryoboxes from Dewar.
<img alt="Figure 6" src="/files/ftp_upload/3903/3903fig6.jpg" /> 
Figure 6. One end of the slotted flat plate is bent using pliers and covered with foam to provide better grip especially for deep Dewars, as shown in this case.
<img alt="Figure 7" src="/files/ftp_upload/3903/3903fig7.jpg" /> 
Figure 7. Strainer with wire mesh and stainless steel handle is used to retrieve cryovials from Dewar. If the handle is not provided with a slot, drill with carbide bit will be required for drilling a hole.
<img alt="Figure 8" src="/files/ftp_upload/3903/3903fig8.jpg" /> 
Figure 8. To ease retrieval of cryovials, strainer is bent along the neck using pliers to get ladle shaped strainer. If strainer is provided with lip as in this case, pliers can be used to bend it. This ensures strainer does not get caught within the Dewar.
<img alt="Figure 9" src="/files/ftp_upload/3903/3903fig9.jpg" /> 
Figure 9. Strainer is secured to the slotted flat plate at one end using hex nut and bolt.
<img alt="Figure 10" src="/files/ftp_upload/3903/3903fig10.jpg" /> 
Figure 10. Sample integrity of total RNA subjected to different sample handling conditions. To explore how RNA quality is affected by sample handling conditions, a set of RNA samples from the same source was subjected to two methods of handlings. Method A consisted of storage of RNA in Dewar subjected to sample retrievals using DIY device, thus no partial defreezing. Method B consisted of storage of RNA in Dewar subjected to repeated partial defreezing by short-term retrievals from the Dewar, thus modeling long term storage with periodic decanting for retrieving fallen samples. Total RNA integrity was then analyzed by gel electrophoresis. Lane1: Sample before storage in Dewar; Lane 2: Sample from Dewar subjected to retrieval by Method A; Lane 3, 4: Sample from Dewar subjected to retrieval by Method B; M: 100 bp ladder.

<ol>
	<li>Kittel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Cryogenic Engineering. , 41 (1996).</li><li>Edeskuty, F. J., Walter, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Stewart Safety in the handling of cryogenic fluids. , (1996).</li><li>Council, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Prudent Practices in the Laboratory: Handling and Management of Chemical Hazards. , (2011).</li><li>Muyal, J., Muyal, V., Kaistha, B., Seifart, C., Fehrenbach, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+comparison+of+RNA+extraction+techniques+from+frozen+and+fresh+lung+tissues:+checkpoint+towards+gene+expression+studies.">Systematic comparison of RNA extraction techniques from frozen and fresh lung tissues: checkpoint towards gene expression studies.</a> Diagnostic Pathology. 4, 9 (2009).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (2001).</li><li>Holland, N. T., Smith, M. T., Eskenazi, B., Bastaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+sample+collection+and+processing+for+molecular+epidemiological+studies.">Biological sample collection and processing for molecular epidemiological studies.</a> Mutation Research/Reviews in Mutation Research. 543, 217-234 (2003).</li><li>Lynch, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CELL,+TISSUE+AND+ORGAN+CULTURE+|+Storage+and+Cryopreservation.">CELL, TISSUE AND ORGAN CULTURE | Storage and Cryopreservation.</a> Encyclopedia of Rose Science. , 106-111 (2003).</li><li>Crawford, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plastics Engineering. , (1998).</li><li>Gorenc, B., Tinyou, R., Syam, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Steel designers' handbook. , (1996).</li><li>Harper, C. A., Petrie, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastics+materials+and+processes:+a+concise+encyclopedia.">Plastics materials and processes: a concise encyclopedia.</a> Wiley-Interscience. , (2003).</li></ol>

No conflicts of interest declared.

One of the most common methods of retrieving cryoboxes and cryovials from the bottom of the Dewar tank is to decant liquid nitrogen into a spare container and pull out the samples remaining in the flask or floating in the decanted liquid nitrogen. This is, however, an unsafe practice that may cause cryogenic burns or asphyxiation due to prolonged exposure to liquid nitrogen vapors2. Other common way of retrieval of fallen cryovials involves use of cryovial tongs3. However, the limited visibility within the dark space of the tank filled with liquid nitrogen restricts their maneuverability.
The Cryotolerant DIY retrieval device described above provides a cheaper, safer and easier alternative for retrieving fallen cryoboxes and cryovials from the bottom of deep flasks with limited visibility. The ease of handling the DIY retrieval device circumvents the need to empty the flasks prior to retrieval. Moreover, the retrieval of cryovial with the help of said device can be carried out by a single individual. Additionally, the use of the DIY retrieval device substantially shortens the amount of time other samples stored in the same unit spend under ambient temperatures, thereby improving sample storage conditions6,7.
Many commonly used materials such as plastic, carbon steel and rubber become brittle in liquid nitrogen, and often fracture under stress8,9,10. The stainless steel or galvanized components used in the DIY device are resistant to damage from sub-freezing temperatures of cryogenic fluids. Furthermore, these components are easily available in local hardware stores and of low cost with the construction of DIY device amounting to less than $50.00. The long handle helps prevent operator from direct contact with the cryogenic fluids and the perforated base and strainer allow instant draining of liquid nitrogen during sample recovery. The rapid retrieval allows minimum exposure to liquid nitrogen vapors making sample retrieval a safer process. Due to its modular design, the DIY retrieval device has the flexibility to be used for either boxes or vials. Further, the simplicity of the design allows the device to be adapted for Dewars of varying shapes and sizes and possibly to work with other types of cryogenic fluids. The use of this device may, however, be limited in the case of cryogenic tanks that do not have a flat bottom.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Hillman Group Plated Slotted Flat 1-3/8 x 4 feet; 14 Gauge</td>
 <td>Local Hardware shop</td>
 <td>N/A</td>
 <td>1 unit required</td>
 </tr>
 <tr>
 <td>Crown Bolt Stainless Steel #6 - 32 x 3/8 in. Oval Head Phillips Drive Machine Screw</td>
 <td>Local Hardware shop</td>
 <td>Model # 42281</td>
 <td>4 units required</td>
 </tr>
 <tr>
 <td>Crown Bolt 3/8 in. x 1-1/2 in. Zinc-Plated Steel Fender Washers</td>
 <td>Local Hardware shop</td>
 <td>Model # 20122</td>
 <td>4 units required</td>
 </tr>
 <tr>
 <td>Crown Bolt Zinc Plated #8-32 Machine Screw Nut</td>
 <td>Local Hardware shop</td>
 <td>Model # 18512</td>
 <td>4 units required</td>
 </tr>
 <tr>
 <td>Simpson Strong-Tie BC40Z 4 In.x4 in. Half Base</td>
 <td>Local Hardware shop</td>
 <td>Model # BC40Z</td>
 <td>2 units required</td>
 </tr>
 <tr>
 <td>Simpson Strong-Tie 6 in. x 6 in. T Strap</td>
 <td>Local Hardware shop</td>
 <td>Model # 66T</td>
 <td>1 unit required</td>
 </tr>
 <tr>
 <td>Oneida 6" Mesh Strainer</td>
 <td>Local Grocery shop</td>
 <td>Model # 7859W</td>
 <td>1 unit required</td>
 </tr>
 <tr>
 <td>The Hillman Group Galvanized Hex Nuts 3/8-16</td>
 <td>Local Hardware shop</td>
 <td>Model #:810509</td>
 <td>6 units required</td>
 </tr>
 <tr>
 <td>The Hillman Group Galvanized Hex Bolts 3/8- 16</td>
 <td>Local Hardware shop</td>
 <td>&nbsp;</td>
 <td>4 units required</td>
 </tr>
 <tr>
 <td>Foam for insulation</td>
 <td>Local Hardware shop</td>
 <td>&nbsp;</td>
 <td>1 unit required</td>
 </tr>
 <tr>
 <td>Screw Driver</td>
 <td>Local Hardware shop</td>
 <td>&nbsp;</td>
 <td>1 unit required</td>
 </tr>
 <tr>
 <td>Pliers</td>
 <td>Local Hardware shop</td>
 <td>&nbsp;</td>
 <td>1 unit required</td>
 </tr>
 </tbody>
</table>

do-it-yourself device for recovery of cryopreserved samples accidentally dropped into cryogenic storage tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Watch this Scientific Journal Video about Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks at JoVE.com

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

do-it-yourself-device-for-recovery-cryopreserved-samples-accidentally

Nanyang Technological University

Research

JoVE Journal

Biology

25.2K Views.  George Mason University. Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Video: Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community&#39;s knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

The study of amphibian biology provides valuable information on the reproductive, physiological, embryological and developmental processes that drive organisms of many taxonomic groups. Here, we present a comprehensive guide on different methodologies that can be used to study ovarian control and monitoring in amphibians.

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive ...

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

Presented here is a protocol for cryogenic transfer of frozen samples into the dynamic nuclear polarization (DNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The protocol includes directions for rotor storage prior to the experiment and directions for viability measurements before and after the experiment.

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.