We thank Nipun Basrur, Adriana K. Rosas Villegas, Nadav Shai, and Trevor Sorrells for comments on the manuscripts, and Zhongyan Gong and Kyrollos Barsoum for technical assistance. We thank Alex Wild for photographs used in Figure 1. K.V. was supported by the Boehringer Ingelheim Fonds PhD fellowship. V.J. was supported in part by NIH T32-MH095246. This work was supported in part by a grant to The Rockefeller University from the Howard Hughes Medical Institute through the James H. Gilliam Fellowships for Advanced Study program to V.J. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. NSF DGE-1325261 to V.J. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

<ol>
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The authors have nothing to disclose.

For many laboratory applications, artificial membrane feeders offer distinct benefits compared to live hosts by allowing researchers the ability to directly manipulate the contents of the meal. Although multiple methods are available for artificial membrane feeding, the method described here offers advantages in flexibility, cost, and throughput. In comparison to other commercial membrane feeders, the Glytube assay requires a small meal volume, making it an efficient delivery mechanism for costly reagents, including drugs or pathogens, by minimizing the total volume required7,35. As both the protein-free saline and artificial blood meals promote engorgement, compounds or pathogens can be added to either meal as a high-throughput and non-invasive alternative to injections. Additionally, each component of the Glytube can easily be washed, replaced, or scaled up to deliver and quantify multiple meal types without cross-contamination of the feeding apparatus.
To quantify meal volumes consumed by mosquitoes, the fluorescence-based method enables more precise meal size quantification than weighing the mosquitoes before and after feeding. It should be noted that this method is an end-point assay. In contrast, weighing allows the mosquitoes to be kept alive for further experimentation. By using a plate reader, the fluorescence-based method can be easily scaled up for high-throughput quantification of meals consumed by hundreds of individual females.
To achieve high feeding rates, a combination of sufficient host cues must be present to activate female host-seeking behavior and attract females to the feeder. If mosquitoes are not crowding underneath the Glytube, the meal may not be properly warmed, or CO2 delivery may not be sufficient. Addition of human odor to the membrane surface reliably increases attractiveness of the artificial membrane. If mosquitoes are observed underneath the Glytube but fail to feed, the meal composition may be at fault. Females may not feed if the meal itself is not warm, the blood is too old, or if the additives to the meal are intrinsically aversive or cause an undesirable chemical reaction36. Additional ATP also reliably increases feeding rates and can be scaled up to a final concentration of 2 mM in each of the recipes provided. Females may not feed if the parafilm is not pulled taut across the Glytube cap; the parafilm should be uniformly transparent and should not buckle, as this prevents the female from being able to effectively pierce the parafilm with her stylet. If the meal leaks through the Glytube onto the mesh, the parafilm may have torn during the stretching process and should be replaced.
Changing the meal composition can also allow researchers to manipulate the length of time needed to clear the meal from the midgut as well as the subsequent host-seeking behavior. The meals presented here require 24&#8211;36 h for digestion7 similar to animal-derived blood. After feeding on any of these meals, females will suppress host-seeking during the digestion time window. Since the saline meal lacks protein, females return to host-seeking after the meal is cleared. If a faster return is desirable, researchers can choose alternate &#8220;quick clearing&#8221; saline meals that are excreted in approximately 6 h27. While the composition of the saline meal presented here is matched to directly compare results with the artificial blood meal, the &#8220;quick clearing&#8221; meal more closely matches physiological salt levels found in vertebrate blood.
The methods described here have limitations that should be considered before selecting the assay that is most suited to the researcher&#8217;s experimental goals. The fluorescein measurements described do not allow mosquitoes to be used again for additional experimentation. However, weight measurements can be taken prior to meal size quantification using the fluorescein assay. If weight and meal size are consistent across multiple trials for a given meal, weight can be used as a proxy in future experiments. Moreover, this protocol does not distinguish between deficits in host-seeking versus blood-feeding behavior; mosquitoes that show impairments in finding the membrane feeder will have a reduction in feeding rates and/or meal size. By adding a camera to record behavior throughout the assay, researchers can determine whether the females cannot find the Glytube, or whether they find the Glytube, but do not feed.
The assay described here can be adapted to explore many outstanding questions related to feeding behavior in mosquitoes. For example, the contribution of specific blood proteins can be explored by altering the ratio of constituent proteins or total protein concentration in the artificial blood meal. To evaluate meal sizes from multiple feeding events, dyes with distinct fluorescence spectra can be added to differentiate meals from unique sources37. This protocol can also be modified to separately stimulate the internal mouthparts that detect blood and that are used for ingestion (i.e., stylet), and the chemosensory appendages that contact skin (i.e., labium, legs) as the mosquito lands to begin blood feeding36. For example, if ligands are added directly to the meal, they do not contact the labium and legs, since the membrane is pierced only by the stylet. If ligands are added to the outer surface of the parafilm instead, they remain separated from the meal and may be contacted by the labium and legs36. Finally, the detailed kinetics of blood-feeding behavior are not well understood and the method presented here could be modified to combine high-resolution tracking with machine learning tools to extract behavioral readouts of locomotion, posture, and feeding dynamics38.
This protocol is aimed at being user-friendly and cost-effective, with the ability to serve researchers employing pharmacological and genetic manipulations to study mosquito blood-feeding and post-blood-feeding behavior.

We present a protocol for feeding modified blood meals to Aedes aegypti mosquitoes using an artificial membrane feeder and precisely measuring the meal volume consumed by each individual mosquito. This protocol can be flexibly adapted to alter the content of the meal or to compare the meal volume consumed by different experimental groups of mosquitoes.
The Ae. aegypti mosquito threatens global health by spreading pathogens that cause diseases including yellow fever, dengue fever, chikungunya, and Zika1,2,3,4,5. Ae. aegypti females are obligate blood-feeders; they must consume vertebrate blood to obtain the necessary protein for egg development, and each clutch of eggs requires a full blood meal from at least one host6,7,8. The female mosquito first bites her host by piercing the skin with her stylet and injecting saliva, which contains compounds that trigger the host&#8217;s immune response9. She then feeds by pumping blood through her stylet into her midgut. While consuming a blood meal from an infected host, she may ingest blood-borne pathogens6,8, which then migrate from the mosquito&#8217;s midgut to her salivary glands10. Female mosquitoes infected in this manner can spread disease by injecting pathogens along with saliva when biting subsequent hosts11,12. Understanding and quantifying the mechanisms of blood-feeding behavior are crucial steps in controlling the transmission of mosquito-borne diseases.
Many laboratory protocols for mosquito rearing and experimentation use live animals including mice, guinea pigs, or humans as a blood source13,14,15,16. The use of live animals imposes ethical concerns as well as complex requirements for personnel training, animal housing and care, and compliance with Institutional Animal Care and Use Committee (IACUC) policies. It also limits the types of compounds that can be delivered to mosquitoes, which constrains the studies that can be carried out17.
Artificial blood-feeding apparatuses, which typically use a membrane system to simulate host skin, are useful tools for studying blood-feeding behaviors that circumvent the need for the maintenance of live hosts. Whole blood can be purchased from a number of vendors and fed to mosquitoes using heated, water-jacketed artificial membrane feeders or similar devices18,19. In this protocol, we demonstrate the use of small, disposable membrane feeders termed &#8220;Glytubes&#8221;. This membrane feeder, previously published by Costa-da-Silva et al. (2013)20, can be easily assembled from standard laboratory equipment, making it ideal for delivering blood meals to moderate numbers of mosquitoes and straightforward to scale up for testing larger groups or multiple meal formulations. The Glytube is an inexpensive and efficient alternative to other commercial artificial feeders, which may require larger meal volumes and are more suitable for batch feeding large groups of mosquitoes on a single meal formulation21.
This protocol includes two sections: preparing/delivering artificial meals and quantifying consumption. In the first section, Glytubes are used as an efficient means to deliver manipulated diets. Whole blood may be substituted with an entirely artificial meal to compare the effects of blood substitutes in lieu of a blood meal. A recipe adapted from Kogan (1990)22 is presented here, although multiple artificial meal formulations have been developed23,24. Furthermore, feeding is a less invasive and less laborious method to introduce pharmacological compounds than injection. Due to the low total volume required for each meal (1&#8211;2 mL), this protocol provides an attractive delivery method to reduce the amounts of expensive reagents. Ae. aegypti females readily consume protein-free meals of saline solution with adenosine 5&#8242;-triphosphate (ATP)25,26, which provides a baseline for measuring the effects of single meal components. For example, Neuropeptide Y-like receptor 7 (NPYLR7) in Ae. aegypti is known to mediate host-seeking suppression after a protein-rich blood meal, and when NPYLR7 agonists are added to a protein-free saline meal, female mosquitoes exhibit host-seeking suppression similar to those that have consumed whole blood7.
In the second section, steps for quantifying the volume of each meal consumed by an individual female mosquito are presented. This assay is fluorescence-based and captures feeding status in higher resolution than methods in which females are classified as &#8220;fed&#8221; or &#8220;unfed&#8221; based on visual assessment of abdominal distension alone. By adding fluorescein to the meal prior to feeding, meal volumes ingested by individuals can be quantified by homogenizing each mosquito in a 96-well plate and measuring fluorescence intensity as a readout. This assay can measure differences in feeding vigor in response to variables such as meal composition or the mosquitoes&#8217; genetic background. Precise quantification is critical for intermediate meal sizes, for example when females are offered suboptimal meals containing feeding deterrents or when they consume sucrose meals of variable sizes27. If fed mosquitoes are required for subsequent behavioral assays after meal size quantification, meal size can instead be calculated by weighing anesthetized females in groups and estimating the average increased mass per individual. Although less precise than fluorescein marking, weighing still provides an aggregated estimate of meal volume and allows examination of the meal&#8217;s effect on downstream processes, such as fecundity or subsequent host attraction. While blood meal size is variable and can be influenced by a myriad of factors11,28,29, ingested meal sizes measured with the methods described here are consistent with previous quantifications7,30,31.

<table>
	<tbody>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-70C</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mm diameter borosilicate solid-glass bead</td>
			<td>MilliporeSigma</td>
			<td>Z143928</td>
			<td>For use for bead mill homogenizer; not required if using pellet pestle grinder</td>
		</tr>
		<tr>
			<td>32 oz. high-density polyethylene (HDPE) plastic cup</td>
			<td>VWR</td>
			<td>89009-668</td>
			<td>Example mosquito container used for feeding assays shown; alternate options can be used</td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-49A</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well black polystyrene plate</td>
			<td>ThermoFisher</td>
			<td>12-566-09</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well PCR plate sealing film</td>
			<td>Bio-Rad</td>
			<td>MSB1001</td>
			<td>Alternate options can be used</td>
		</tr>
		<tr>
			<td>96-well PCR plates</td>
			<td>Bio-Rad</td>
			<td>HSP9621</td>
			<td>Alternate options can be used</td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate (ATP) disodium salt hydrate</td>
			<td>MilliporeSigma</td>
			<td>A6419</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin (human serum)</td>
			<td>MilliporeSigma</td>
			<td>A9511</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Fisher Scientific</td>
			<td>01-213</td>
			<td>Alternate options can be used to block light entering fluorescein container</td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Fisher Scientific</td>
			<td>01-911</td>
			<td>Alternate options can be used</td>
		</tr>
		<tr>
			<td>Bead mill homogenizer</td>
			<td>Qiagen</td>
			<td>85300</td>
			<td>Not required if using pellet pestle grinder</td>
		</tr>
		<tr>
			<td>Cotton ball</td>
			<td>Fisher Scientific</td>
			<td>22456880</td>
			<td>For nectar-feeding; alternate options can be used</td>
		</tr>
		<tr>
			<td>Defibrinated sheep blood</td>
			<td>Hemostat Laboratories</td>
			<td>DSB100</td>
			<td>Alternate options can be used</td>
		</tr>
		<tr>
			<td>Drosophila CO2 fly pad</td>
			<td>Tritech Research</td>
			<td>MINJ-DROS-FP</td>
			<td>Alternate options can be used</td>
		</tr>
		<tr>
			<td>Fluorescein</td>
			<td>MilliporeSigma</td>
			<td>F6377</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence plate-reader</td>
			<td>ThermoFisher</td>
			<td>VL0000D0</td>
			<td>Alternate options can be used</td>
		</tr>
		<tr>
			<td>Gamma-globulin (human blood)</td>
			<td>MilliporeSigma</td>
			<td>H7379</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>MilliporeSigma</td>
			<td>G7893</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemoglobin (human)</td>
			<td>MilliporeSigma</td>
			<td>G4386</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory wrapping film - parafilm</td>
			<td>Fisher Scientific</td>
			<td>13-374</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>Fisher Scientific</td>
			<td>90-691</td>
			<td>Alternate magnetic stirrers can be used</td>
		</tr>
		<tr>
			<td>Microcentrifuge for 96-well plate</td>
			<td>VWR</td>
			<td>80094-180</td>
			<td>Alternate options can be used</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes</td>
			<td>MilliporeSigma</td>
			<td>2236412</td>
			<td>Alternate options can be used</td>
		</tr>
		<tr>
			<td>Pellet pestle grinder</td>
			<td>VWR</td>
			<td>KT749521-1500</td>
			<td>Not required if using bead mill homogenizer</td>
		</tr>
		<tr>
			<td>Phosphate buffered solution (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BW17-516F</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td>Alternate options can be used, such as a lathe for better consistency of cutting</td>
		</tr>
		<tr>
			<td>Rubber bands</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone tubing</td>
			<td>McMaster Carr</td>
			<td></td>
			<td>Needed if using a fly pad for CO2 delivery</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Fisher Scientific</td>
			<td>S233</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>MilliporeSigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir bars</td>
			<td>Fisher Scientific</td>
			<td>14-512</td>
			<td>Alternate magnetic stir bars can be used</td>
		</tr>
		<tr>
			<td>Translucent polypropylene storage box with removable lid</td>
			<td></td>
			<td></td>
			<td>Example box used for feeding assays shown</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td>Alternate heating device may be used</td>
		</tr>
		<tr>
			<td>White 0.8 mm polyester mosquito netting</td>
			<td>American Home &#38; Habit Inc.</td>
			<td>F03A-PONO-MOSQ-M008-WT</td>
			<td>Alternate options can be used</td>
		</tr>
	</tbody>
</table>

feeding and quantifying animal-derived blood and artificial meals in aedes aegypti mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Figure 1 presents a schematic for assembling the Glytube, whereas Figure 2 shows an overview of the experimental design to measure meal size using the fluorescence-based assay described here. Figure 3 provides representative fluorescein meal size measurements from a blood-feeding experiment. Figure 4, Figure 5, and Figure 6 illustrate a sampling of biological questions that can be addressed using this protocol. Applications of the protocol are wide-ranging and include altering blood meal composition, feeding pharmacological compounds, precisely quantifying sub-optimal blood meals or smaller nectar meals, and comparing feeding behavior across mosquito genotypes.
To generate a standard curve for meal volume calculations, fluorescence readings are plotted from the designated reference wells each containing an unfed mosquito and a known volume of the meal with 0.002% fluorescein (Figure 3A). Fluorescence readings from the remaining wells, which contain mosquitoes from either the negative control group of unfed mosquitoes or the experimental group of mosquitoes offered a meal, are compared to this standard curve to quantify the meal volume (&#181;L) consumed by each mosquito (Figure 3B). To validate the baseline readings in this assay, it should be confirmed that mosquitoes from the unfed negative control group are not assigned a positive value of &#181;L consumed (Figure 3B, left). Although all females in the experimental group were offered the blood meal, some mosquitoes fed (Figure 3B, middle) and some did not (Figure 3B, right). This result demonstrates that two types of data can be obtained from this protocol: 1) the percentage of total females that feed on a given meal, and 2) the volume ingested by the females that feed on a given meal.
This protocol can be used to deliver and quantify meals with various protein compositions. Figure 4A,B show data collected using meals with added fluorescein. The proportion of mosquitoes that fed and the meal volume they ingested, respectively, were calculated from the fluorescence readings. These readings are highly sensitive and allow for precise quantification of &#181;L, but have the limitation that mosquitoes cannot be used for future live experiments. Figure 4C,D show data collected from an independent experiment with mosquitoes that were scored as fed or unfed by eye after they were offered meals without fluorescein. Meal size was calculated as average weight/female from groups of 5 mosquitoes. Although these weight measurements are less sensitive than fluorescence measurements, they allow the females to be recovered and used for further live experimentation. The proportion of mosquitoes that feed can vary across different experimental days, as reflected in Figure 4A and Figure 4C.
Figure 5 shows the volume consumed of meals containing drugs that regulate mosquito host-seeking behavior. In these experiments, females were offered blood, saline + ATP, or saline + ATP&#160;meals with 100 &#956;M of the human NPY Y2 receptor agonist, TM30338. This drug alters host-seeking behavior through activation of Ae. aegypti NPY-like receptor 7. Measuring meal sizes is critical for the interpretation of experiments to assess the effect of this drug on post-blood-feeding behavior because it allows the researcher to calculate the dose consumed by each female.
In the previous examples, females were fed either blood or substitute blood meals, all of which resulted in 3&#8211;5 &#181;L meals (Figure 3, Figure 4, Figure 5). This fluorescence-based assay can also be used to measure smaller and/or more variable meal sizes that cannot be accurately discerned from average group weight measurements. In Figure 6, the same fluorescence quantification protocol was used to measure nectar-feeding behavior by exchanging the Glytube for a cotton ball saturated with 10% sucrose containing 0.002% fluorescein. Nectar sugars cannot be presented in the Glytube assay because females cannot detect the presence of nectar sugars with the stylet and do not initiate feeding27. These data allow the researcher to determine that sugar meals are consistently smaller than blood meals, in agreement with previous work34 (Figure 6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61835/61835fig1v2.png" /> 
Figure 1: Setup of Glytube method used to feed meals to mosquitoes.&#160;(A) Schematic of a deconstructed Glytube used to feed blood and other meals to mosquitoes. (B) Schematic of a Glytube presented atop a container of mosquitoes with a mesh lid. Female mosquitoes can pierce through the mesh lid to feed. (C) Photographs of the Glytube (top), and female Aedes aegypti mosquitoes before, during, and after feeding (bottom, from left to right) on a Glytube-delivered meal. Mosquitoes are shown piercing through the mesh covering their container to access the membrane feeder. (D) Photographs showing the appearance of female Ae. aegypti mosquitoes that are unfed (left) and that have engorged on either an artificial blood meal (right, top) or a saline + ATP meal (right, bottom). The Glytube method was previously published in Costa-da-Silva et al. (2013)20. Photographs in (C) and (D) are courtesy of Alex Wild. <a href="https://www.jove.com/files/ftp_upload/61835/61835fig1v2large.png" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61835/61835fig2v4.png" /> 
Figure 2: Schematic of how to quantify meal size after Glytube blood-feeding protocol.&#160;(A) Mosquitoes are offered a meal with fluorescein (top, experimental group) or no meal (bottom, unfed negative control group). (B) Individual mosquitoes are added to a 96-well plate after terminating the feeding experiment. (C) Standard curve is generated using known amounts of meal containing 0.002% fluorescein. (D) Mosquitoes are homogenized to release any consumed fluorescein, and fluorescence levels in each well are quantified using a plate reader. This fluorescence quantification method is modified from Liesch et al. (2013)34. <a href="https://www.jove.com/files/ftp_upload/61835/61835fig2v4large.png" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61835/61835fig3v2.png" /> 
Figure 3: Glytube blood-feeding experiment with fluorescein-based quantification.&#160;(A) Standard curve measurements obtained from the wells where a mosquito from the unfed control group was added to a known quantity of meal containing 0.002% fluorescein (y-axis scale = arbitrary units). (B) Meal volume calculated using fluorescence readings for females in the unfed control group (left, black, n = 40), the experimental group that fed on blood (middle, red, n = 37), and the experimental group that did not feed on blood (right, red, n = 23). Each point represents a measurement from an individual female. Data are shown as median with range. Letters indicate statistically distinct groups, Kruskal-Wallis test with Dunn&#8217;s multiple comparison, p&#60;0.01. These data were published in Jov&#233; et al. (2020)27. <a href="https://www.jove.com/files/ftp_upload/61835/61835fig3v5large.png" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61835/61835fig4v2.png" /> 
Figure 4: Quantification of meals with differing protein composition.&#160;Females were offered meals of either sheep blood (red), artificial blood with human blood proteins (Kogan (1990)22) (orange), or protein-free saline + ATP meal (aqua)7. (A) Percentage of females fed scored using fluorescence readings. Each point represents a group of 12&#8211;16 females. Data are shown as medians with ranges, n = 12. (B) Meal volume calculated using fluorescence readings. Each point represents a measurement from an individual female in a single trial from Figure 4A. Data are shown as medians with ranges, n = 12. (C) Percentage of females fully engorged after artificial membrane feeding, scored by eye. Each point represents the percent of females engorged from groups of 20&#8211;30 females. Data are shown as medians with ranges, n = 23. (D) Meal sizes scored as weight/female after feeding status was scored by eye. Weights were calculated as the average of groups of 5 mosquitoes. Data are shown as medians with ranges, n = 23. A&#8211;D: Letters indicate statistically distinct groups, Kruskal-Wallis test with Dunn&#8217;s multiple comparison, p&#60;0.05. <a href="https://www.jove.com/files/ftp_upload/61835/61835fig4v5large.png" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61835/61835fig5v2.png" /> 
Figure 5: Quantification of meals with pharmacological compounds.&#160;Females consume meals of the same size of sheep blood (red), saline + ATP (aqua), and saline + ATP + 100 &#181;M dose of human NPY Y2 receptor agonist TM30338 (dark blue). Meal volume calculated using fluorescence readings. Each point represents a measurement from an individual female. Data are shown as medians with ranges, n = 12. Letters indicate statistically distinct groups, Kruskal-Wallis test with Dunn&#8217;s multiple comparison, p&#60;0.05. <a href="https://www.jove.com/files/ftp_upload/61835/61835fig5v5large.png" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61835/61835fig6v2.png" /> 
Figure 6: Quantification of smaller nectar meals.&#160;(A) Schematic of nectar-feeding assay. (B) Meal volume calculated using fluorescence readings for wild-type females offered meals of either water (blue, n = 36) or 10% sucrose (green, n = 53), each with 0.002% fluorescein, in the nectar-feeding assay. Each point represents a measurement from an individual female. Data are shown as medians with ranges. Letters indicate statistically distinct groups, Mann-Whitney test, p&#60;0.05. These data were published in Jov&#233; et al. (2020)27. <a href="https://www.jove.com/files/ftp_upload/61835/61835fig6v5large.png" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Artificial Blood Meal</td>
		</tr>
		<tr>
			<td></td>
			<td>Concentration of Stock Solution (mg/mL)</td>
			<td>Volume of Stock Solution in Meal (&#956;L/mL)</td>
			<td>Final Meal Concentration (mg/mL)</td>
		</tr>
		<tr>
			<td colspan="4">Protein Components*</td>
		</tr>
		<tr>
			<td>&#947;-Globulins</td>
			<td>50</td>
			<td>300</td>
			<td>15</td>
		</tr>
		<tr>
			<td>Hemoglobin</td>
			<td>35</td>
			<td>230</td>
			<td>8</td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>300</td>
			<td>340</td>
			<td>102</td>
		</tr>
		<tr>
			<td>Total Protein</td>
			<td>-</td>
			<td>-</td>
			<td>125</td>
		</tr>
		<tr>
			<td colspan="4">Non-Protein Components</td>
		</tr>
		<tr>
			<td></td>
			<td>Concentration of Stock Solution (mM)</td>
			<td>Volume of Stock Solution in Meal (&#956;L/mL)</td>
			<td>Final Meal Concentration (mM)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>In &#947;-globulin stock</td>
			<td>-</td>
			<td>5-10</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>In &#947;-globulin stock</td>
			<td>-</td>
			<td>120</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>200</td>
			<td>5</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>-</td>
			<td>125</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">*Protein components are prepared in stock solution of double-distilled water, except for &#947;-Globulins, which are dissolved in 400 mM NaHCO3 and include a variable amount of NaCl (2-4%) in the product.</td>
		</tr>
	</tbody>
</table>
Table 1: Recipe for preparing artificial blood meals (adapted from Kogan (1990)22). Artificial blood consists of protein and non-protein components regularly found in human blood and provides the option to vary the ratios of these components. Mosquitoes can produce eggs after feeding on artificial blood7,22.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Saline Meal</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Concentration of Stock Solution (mM)</td>
			<td>Volume of Stock Solution in Meal (&#956;L/mL)</td>
			<td>Final Meal Concentration (mM)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>400</td>
			<td>300</td>
			<td>120</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>200</td>
			<td>5</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>-</td>
			<td>695</td>
			<td>-</td>
		</tr>
	</tbody>
</table>
Table 2: Recipe for saline meal with ATP (adapted from Duvall et al. (2019)7).&#160;Protein-free saline meals can be used to deliver compounds of interest to mosquitoes while still mimicking the abdominal distension that occurs after blood-feeding, but without triggering the egg development that occurs when proteins are ingested.

Watch this Scientific Journal Video about Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes at JoVE.com

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...

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7.6K Views.  The Rockefeller University. The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

Video: Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).