Protocols involving invertebrate animals are exempt from Instituto Butantan&#39;s Committee on the Use and Care of Animals&#39; approval. However, the maintenance of scorpions described here follows ethical parameters, and animal welfare is respected according to the needs of the species.
1. Housing
<ol>
	<li>Keep the scorpions in communal polypropylene containers as enclosures (height 35 cm, width 35.5 cm, length 72 cm) containing a maximum of 350 animals.</li>
	<li>Maintain the room where the animal enclosures are housed under a controlled temperature of 24 &#176;C (&#177;0.5 &#176;C) in a light cycle of 10 h with lights on and 14 h with lights off.</li>
	<li>Line the enclosure floor (bottom) of each container with kraft paper fixed with masking tape on the edges on all sides so that the animals cannot get under the paper (Figure 3A).</li>
	<li>Use four cardboard trays (egg trays) as vertical substrates, stacked on one side of the container, with a rigid cardboard sheet placed between each tray (Figure 3B,C).</li>
	<li>Place a shallow polypropylene tray (water tray) on the opposite side of the enclosure, containing soaked cotton as a water source for the animals (Figure 3B).</li>
	<li>Keep the containers without lids to increase the airflow. Place a strip of approximately 10 cm of self-adhesive plastic tape on the top of the internal surface of the enclosure and on the upper corners to make the walls smooth enough to prevent the animals from escaping from the container (Figure 3A).</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65737/65737fig03.jpg" /> 
Figure 3: Housing and enclosure preparation. (A) Arrows indicate the kraft paper on the bottom, fixed with masking tape on the edges. Dotted lines indicate the self-adhesive plastic tape placed on the top of the internal surface of the enclosure.&#160;(B)&#160;Vertical substrate (egg trays piled on one side of the container) and a shallow polypropylene tray (water tray) placed on the opposite side. (C)&#160;Vertical substrate detail: egg trays pilled with a rigid cardboard sheet in between two trays. (D)&#160;Cardboard sheets and egg trays positioned in layers, simulating the natural dark and humid habitat of T. serrulatus. (E) Animals accommodated in the substrate. <a href="https://www.jove.com/files/ftp_upload/65737/65737fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Hygiene routine
NOTE: The hygiene routine is divided into two maintenance procedures: complete and partial maintenance. The first one is performed 2 days after feeding the animals or when the enclosure is selected for venom extraction. Partial maintenance is performed when the animals are not fed.
<ol>
	<li>Complete maintenance
	<ol>
		<li>Prepare a clean enclosure with new substrate (egg trays, cardboard, and kraft paper) and a clean water tray, as described in section 1.</li>
		<li>Utilizing long forceps, remove the water tray from the enclosure. If there are any scorpions on the water tray, remove them and place them in the clean enclosure.</li>
		<li>Repeat the same procedure described in step 2.1.2 with all egg trays and cardboard sheets, removing all the scorpions and placing them in the clean enclosure.</li>
		<li>After the removal of the trays, remove all the scorpions left on the floor of the enclosure and place them in the clean enclosure. Be sure to account for any carcasses and discard them properly. Collect all remaining live prey (insects) and remove them from the enclosure.</li>
		<li>Remove all the disposable substrates from the enclosure (egg trays, cardboard sheet, and kraft paper) and properly discard them. Send the reusable items (water tray and polypropylene container) for sanitation. 
		NOTE: The sanitation of the reusable items is performed with 0.5% hypochlorite combined with a neutral detergent. It is mandatory that the materials are thoroughly and abundantly rinsed with running water after using any sanitation product or solution.</li>
	</ol>
	</li>
	<li>Partial maintenance 
	NOTE: Partial maintenance consists of inspecting all the substrates (egg trays and kraft paper) with the aim of removing dead scorpion carcasses and other undesirable debris, but without discarding the substrates or changing the enclosure.
	<ol>
		<li>Perform the inspection using long forceps to gently lift each egg tray and remove the waste.</li>
		<li>If needed, replace one or more egg trays with a clean one.</li>
		<li>Completely replace the water tray with a clean one using long forceps. Transfer any scorpions on the water tray to the egg tray in another part of the enclosure.</li>
	</ol>
	</li>
</ol>
3. Feeding
<ol>
	<li>Feed the animals every 15 days according to the venom extraction schedule. Feed animals that will be milked 7 days prior and 7 days after the venom extraction.</li>
	<li>For the feeding, supply cockroaches (Phoetalia pallida) and crickets (Gryllus sp.) alternately.</li>
	<li>Calculate the amount of food according to the energy intake of the animals; offer 1 prey for every 3 or 5 scorpions in the enclosure.</li>
	<li>Count the calculated amount of prey items and place them alive inside the enclosure, leaving them for 2 days prior to the complete maintenance (section 2.1).</li>
	<li>When offering cockroaches, apply a calcium carbonate with alcohol solution around the top of the enclosures. The solution causes the cockroaches to slip and not be able to climb the side surface of the enclosure, thus preventing the insects from escaping.</li>
</ol>
4. Venom extraction
NOTE: The animals used in the venom extraction routine are adults or pre-adults. Due to the difficulty in determining the complete sexual maturity of the Brazilian yellow scorpion (adult phase), it was determined that animals with a total body length of 5 to 7 cm are eligible for the procedure.
<ol>
	<li>Milk each animal every 2-3 months.</li>
	<li>On the day of venom extraction, count all alive animals in the enclosure and transfer them to a glass container utilizing long forceps (Figure 4).</li>
	<li>Prepare and arrange all the materials and equipment to be used on the top of the airflow cabinet work surface. Plug the electrostimulator power cord into the outlet and turn the electrostimulator and the airflow cabinet on (Figure 2B).
	<ol>
		<li>Use the following electro stimulator parameters settings: potential (difference of the potential between the electrodes): 540 mV, stress (duration of the electric pulse): 2 ms, action (intermittency to moderate the potency used, which would reduce the heating of the telson organic tissue): 70%, cycle (adjust the cyclic repetition): 1 s, and intensity (electric current that circulates through the animal&#39;s muscle): 0.75 mA.</li>
	</ol>
	</li>
	<li>Manually restrain each animal in a way that the telson stands still, following the steps:
	<ol>
		<li>Hold the metassoma of the animal with straight forceps (Figure 5A).</li>
		<li>Immobilize the telson with curved forceps (Figure 5B).</li>
		<li>Restrain the telson with the hand (Figure 5C,D).</li>
	</ol>
	</li>
	<li>After restraint, place the telson in direct contact with the electrostimulator conductor, without the need to use a conductive solution. The electric shock causes an involuntary muscle contraction of the venom gland and consequent ejection of the venom.</li>
	<li>Collect the venom drops directly into a plastic microtube placed close to the electrostimulator conductor (Figure 6).</li>
	<li>Next, release the animal back into the glass container, following the steps:
	<ol>
		<li>Transfer the animal&#39;s telson from the hand to the curved forceps.</li>
		<li>Place the animal gently on the bottom of the glass container.</li>
	</ol>
	</li>
	<li>Perform steps 4.4-4.7 with each animal in the glass recipient.</li>
	<li>After all the animals in the glass recipient have been milked, place them back into the enclosure.</li>
	<li>Store the collected venom at -20 &#176;C until the lyophilization process.</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65737/65737fig04.jpg" /> 
Figure 4: Scorpions in the glass container. <a href="https://www.jove.com/files/ftp_upload/65737/65737fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65737/65737fig05.jpg" /> 
Figure 5: Restraining procedure for venom extraction. (A) Metassoma restrained with straight forceps (black arrow). (B) Telson immobilized with curved forceps (black arrow). (C) Transference of the restrained telson from the forceps to the hand. (D) Telson restrained by the hand. It is recommended that the animals be handled with bare hands since when using gloves, the scorpions can grab the material with their claws, making it difficult to release them at the end of the procedure and considerably increasing the risk of accidents. <a href="https://www.jove.com/files/ftp_upload/65737/65737fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65737/65737fig06.jpg" /> 
Figure 6: Venom extraction. A plastic microtube (white arrow) placed close to the electrostimulator (yellow arrow). The venom drops are collected directly into the tube maintained at room temperature during the procedure. <a href="https://www.jove.com/files/ftp_upload/65737/65737fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Rearing of Brazilian Yellow Scorpion for Venom Extraction

<ol>
	<li>Polis, G. 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> The biology of scorpions. , (1990).</li><li>Lacerda, A. B., 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=Scorpion+envenomation+in+the+state+of+S&#227;o+Paulo,+Brazil:+Spatiotemporal+analysis+of+a+growing+public+health+concern.">Scorpion envenomation in the state of S&#227;o Paulo, Brazil: Spatiotemporal analysis of a growing public health concern.</a> PLoS One. 17 (4), 0266138 (2022).</li><li>Reckziegel, G. C., Pinto, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scorpionism+in+Brazil+in+the+years+2000+to+2012.">Scorpionism in Brazil in the years 2000 to 2012.</a> Journal of Venomous Animals and Toxins Including Tropical Diseases. 20, 46 (2014).</li><li>Guerra-Duarte, C., 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=Scorpion+envenomation+in+Brazil:+Current+scenario+and+perspectives+for+containing+an+increasing+health+problem.">Scorpion envenomation in Brazil: Current scenario and perspectives for containing an increasing health problem.</a> PLoS Neglected Tropical Diseases. 17 (2), 0011069 (2023).</li><li>Pimenta, R. J. G., 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=Selected+to+survive+and+kill:+Tityus+serrulatus,+the+Brazilian+yellow+scorpion.">Selected to survive and kill: Tityus serrulatus, the Brazilian yellow scorpion.</a> PLoS One. 14 (4), e0214075 (2019).</li><li>Lacerda, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+areas+vulnerable+to+scorpionism+and+its+association+with+environmental+factors+in+S&#227;o+Paulo,+Brazil.">Detection of areas vulnerable to scorpionism and its association with environmental factors in S&#227;o Paulo, Brazil.</a> Acta Tropica. 230, 106390 (2022).</li><li>Torrez, P. P. Q., Dourado, F. S., Bertani, R., Cupo, P., Fran&#231;a, F. O. d. e. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scorpionism+in+Brazil:+exponential+growth+of+accidents+and+deaths+from+scorpion+stings.">Scorpionism in Brazil: exponential growth of accidents and deaths from scorpion stings.</a> Journal of the Brazilian Society of Tropical Medicine. 52, 20180350 (2019).</li><li>Amado, T. 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=Vulnerable+areas+to+accidents+with+scorpions+in+Brazil.">Vulnerable areas to accidents with scorpions in Brazil.</a> Tropical Medicine &amp; International Health. 26 (5), 13561 (2021).</li><li>Brasil, Minist&#233;rioda da Sa&#250;de, Funda&#231;&#227;o Nacional de Sa&#250;de. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual de diagn&#243;stico e tratamento de acidentes por animais pe&#231;onhentos. , (2001).</li><li>Candido, D. M., Lucas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+scorpions+of+the+genus+Tityus+Koch+(Scorpiones,+Buthidae)+for+venom+obtention+at+Instituto+Butantan,+S&#227;o+Paulo,+Brazil.">Maintenance of scorpions of the genus Tityus Koch (Scorpiones, Buthidae) for venom obtention at Instituto Butantan, S&#227;o Paulo, Brazil.</a> Journal of Venomous Animals and Toxins including Tropical Diseases. 10 (1), 000100007 (2004).</li><li>Lucas, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laborat&#243;rio+de+Artr&#243;podes+do+Instituto+Butantan+e+os+aracn&#237;deos+pe&#231;onhentos.">Laborat&#243;rio de Artr&#243;podes do Instituto Butantan e os aracn&#237;deos pe&#231;onhentos.</a> Hist&#243;ria Ci&#234;ncias Sa&#250;de Manguinhos. 10 (3), 000300011 (2003).</li><li>Braga-Pereira, G. F., Santos, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asexual+reproduction+in+a+sexual+population+of+the+Brazilian+yellow+scorpion+(Tityus+serrulatus,+Buthidae)+as+evidence+of+facultative+parthenogenesis.">Asexual reproduction in a sexual population of the Brazilian yellow scorpion (Tityus serrulatus, Buthidae) as evidence of facultative parthenogenesis.</a> The Journal of Arachnology. 49 (2), (2021).</li></ol>

The authors have no conflicts of interest to disclose.

The application of the methods described allows us to keep a large number of T. serrulatus individuals and gives us consistent predictability of the number of scorpions needed for annual venom production. This way, we are able to provide enough batches of venom in advance to supply the antivenom manufacturing process. At the same time, the development of pre-established schedules for maintenance, feeding, and venom extraction is an essential part of the activities and assists in complying with the described prot...

Scorpions are arthropods belonging to the class Arachnida, order Scorpiones, which comprises approximately 2,621 species1,2. These animals have a wide geographical range and are present on all continents except Antarctica1. Envenomation caused by scorpions results in the morbidity or death of thousands of people every year globally3. In 2019, it was estimated that there were more than 1.2 million accidents and 3,500 annual deaths caused by these animals. In Brazil, the number of cases has been increasing exponentially, reaching more than 100,000 cases per year since 20174,5,6. The uncontrolled urbanization observed in Brazil in the past decades, without adequate sewage treatment and regular collection and disposal of garbage, associated with environmental degradation and climate changes, has provided conditions for the proliferation of invasive scorpions, such as T. serrulatus, increasing the contact with humans and thus resulting in harmful accidents4,7,8. There are about 178 scorpion species in Brazil, but stings of medical importance are caused by the genus Tityus, with four species (T. serrulatus, T. bahiensis, T. stigmurus, and T. obscurus) being of medical concern, with Tityus serrulatus being responsible for the most severe cases and deaths7,9.
Instituto Butantan is the main antivenom-manufacturing laboratory in Brazil, providing approximately 70,000 vials of scorpion antivenom for the Brazilian health system. Briefly, the steps involved in antivenom manufacturing include the inoculation of venom-derived antigens in equines, the collection and purification of rich-antibodies plasma, resulting in 5 mL vials of scorpion antivenom. Each vial is capable of neutralizing, at least, 1 mg of scorpion venom per mL of antivenom. The Arthropods Bioterium is an integral part of the industrial center, responsible for supplying the source material for the scorpion antivenom.
The Arthropods Bioterium at Instituto Butantan was originally named the Laboratory of Arthropods and was established in 1967. However, it was only in 1995 that the Laboratory moved to an exclusive facility dedicated to housing venomous arthropods10,11. At that time, the scorpions were kept in a room of 12 m2 with no controlled temperature and distributed among approximately 13 enclosures, each housing a maximum of 300 animals10. Due to the increasing demand for venom over the years and the consequent need to increase the number of animals, along with improvements in the venom extraction process, a major change occurred in 2016. The Laboratory was integrated into the industrial center of Instituto Butantan and was renamed the Arthropods Bioterium. Additionally, in 2016, the Bioterium moved to a new purpose-built facility. The new scorpion room covers an area of 24 m2 and contains approximately 48 polypropylene enclosures, each housing up to 300 animals, as shown in Figure 1. This results in a total of 10,000 to 20,000 individuals being maintained under controlled captive conditions, with variations throughout the year. The scorpions are carefully managed under strict husbandry protocols to ensure their welfare while meeting the high production demands and adhering to good practices and ethical standards for animal care. For venom extraction, a specific room was designed and constructed, housing two airflow cabinets as shown in Figure 2A. These cabinets serve as safety measures to prevent technicians from inhaling venom particles during the extraction procedure. Personal protective equipment (PPE) is mandatory for the technicians, including synthetic aprons and FFP2 masks, to avoid any contamination and ensure their safety.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65737/65737fig01v2.jpg" /> 
Figure 1: General view of the scorpion&#39;s room of the Arthropods Bioterium. <a href="https://www.jove.com/files/ftp_upload/65737/65737fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65737/65737fig02.jpg" /> 
Figure 2: Working area. (A) General view of the venom extraction room of the Arthropods Bioterium. (B) Airflow cabinet work surface. All the materials and equipment are prepared and arranged on the top of the airflow cabinet work surface. The black arrow indicates the electrostimulator device, and the red arrow indicates the forceps. <a href="https://www.jove.com/files/ftp_upload/65737/65737fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
About 90% of the T. serrulatus scorpions in the Arthropods Bioterium originate from different sites, collected by municipal health agents as part of a surveillance and control program for these animals. The scorpions are caught in urban areas and then sent to Instituto Butantan as part of a collaborative program. T. serrulatus scorpions are parthenogenetic, meaning that birth occurs as a consequence of the development of offspring from unfertilized eggs, without the presence of an individual of the opposite sex12. Due to this type of reproduction, some animals are born in captivity in our facility and are maintained until they reach the adult phase, following a similar husbandry protocol as described below. Once they reach the adult stage, they are added to the venom extraction routine. Upon arrival, the scorpions undergo initial screening, and those in good health conditions are kept in collective enclosures containing a maximum of 350 individuals, divided by their origin. For each enclosure, a control form is filled out daily, providing data on the date of feeding and the type of prey offered (cricket or cockroach), water supply, cleaning procedures, the number of dead animals, and the number of remaining live ones inside the enclosure.
In a pre-scheduled agenda, animals are subjected to electric stimulation, and the resulting venom of hundreds of individuals is then lyophilized, resulting in batches of freeze-dried venom. The standardized venom obtained from this process is destined for horse immunization as part of the manufacturing process, as well as to provide reference material for quality control of the active substance and the final product.
There are very few facilities in the world capable of keeping such a large number of scorpions and performing the volume of venom extractions in accordance with good manufacturing practices and ethical use of the animals, as done in the Arthropods Bioterium of Instituto Butantan. Therefore, our objective is to describe the scorpion maintenance protocols and venom extraction procedures used in T. serrulatus husbandry, which successfully provide the necessary amount of venom for the production of scorpion antivenom.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Carboard sheet</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg tray</td>
			<td>NA</td>
			<td>NA</td>
			<td>cardboard 36 places egg trays</td>
		</tr>
		<tr>
			<td>Electro stimulator device</td>
			<td>Instituto Butantan</td>
			<td>ART-FV-01</td>
			<td>Internally designed for the venom extraction&#160;</td>
		</tr>
		<tr>
			<td>Kraft paper</td>
			<td>NA</td>
			<td>NA</td>
			<td>regular kraft paper</td>
		</tr>
		<tr>
			<td>Making tape</td>
			<td>NA</td>
			<td>NA</td>
			<td>White masking tape (24 mm x 50 m)</td>
		</tr>
		<tr>
			<td>Polyproplene container</td>
			<td>NA</td>
			<td>NA</td>
			<td>70 L Polypropylene box (H &#8211; 35 cm, W &#8211; 35.5 cm, L - 72 cm)</td>
		</tr>
		<tr>
			<td>Polyproplene tray</td>
			<td>NA</td>
			<td>NA</td>
			<td>Polypropylene tray (H &#8211; 3.0 cm, W &#8211; 18.5 cm, L - 30 cm)</td>
		</tr>
		<tr>
			<td>Self-adhesive plastic</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

captive maintenance and venom extraction of tityus serrulatus (brazilian yellow scorpion) for antivenom production

Scorpion envenomation is a public health problem in several tropical and subtropical countries. Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow scorpion) are responsible for approximately 150,000 envenoming cases per year in Brazil, of which 10% require antivenom treatment to reverse life-threatening venom effects. Therefore, thousands of T. serrulatus individuals are maintained under controlled captivity conditions for venom extraction, subsequently used in the production of the national supply of scorpion antivenom. Instituto Butantan is the main antivenom-manufacturing laboratory in Brazil, providing about 70,000 vials of scorpion antivenom for the Brazilian health system. Thus, the husbandry protocols and venom extraction methodologies are key points for the success of large-scale, standardized venom production. The objective of this article is to describe the captivity protocols of T. serrulatus husbandry, encompassing the husbandry routine and the venom extraction procedures, following good manufacturing practices, and ensuring animal welfare. These practices allow for the maintenance of up to 20,000 animals in captivity, with a routine of 3,000 to 5,000 scorpions milked monthly according to antivenom manufacturing demand, achieving an average of 90% of positive extraction.

Thirty standard operational procedures (SOPs) were developed for each procedure, ensuring the repeatability of the procedures among technicians and compliance with quality parameters. The average mortality rate after the extraction procedure is around 12%, which may be considered low, considering the electrostimulation routine and the captivity environment, where stress factors are successfully reduced by applying the procedures.
To keep the animals healthy, a tray covered with cotton and wate...

Watch this Scientific Journal Video about Optimizing Scorpion Venom Extraction for Antivenom Production at JoVE.com

Author Spotlight: Optimizing Scorpion Venom Extraction for Antivenom Production 

Here, we present successful maintenance and venom extraction protocols for large-scale Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow scorpion) husbandry, with the aim of providing venom for subsequent scorpion antivenom production to meet the demand of the Brazilian health system.

Captive Maintenance and Venom Extraction of Tityus serrulatus (Brazilian Yellow Scorpion) for Antivenom Production

Scorpion envenomation is a public health problem in several tropical and subtropical countries. Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow ...

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2.0K Views.  Instituto Butantan. Here, we present successful maintenance and venom extraction protocols for large-scale Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow scorpion) husbandry, with the aim of providing venom for subsequent scorpion antivenom production to meet the demand of the Brazilian health system.

Video: Author Spotlight: Optimizing Scorpion Venom Extraction for Antivenom Production 

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

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Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune diseases and cancer2. Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes 1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays 6. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

A simple protocol for preparing extracts of human tissue to be used as a source of antigens in functional T-cell assays is described. This method allows T-cell responses to tissue-derived antigens to be measured in vitro.

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.

This article provides a protocol for the extraction of venom from spiders using electrical stimulation in order to 1) conduct proteomic characterization, 2) stimulate venom gland gene expression, and 3) perform functional studies of venoms. This is followed by a description of venom gland microdissections for gene expression studies.

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...