This work was supported by National Natural Science Foundation of China (No. 31872287), Natural Science Foundation of Jiangsu Province (NO. BK20181456) and Six talent peaks project in Jiangsu Province (No. SWYY-146).

1. Maintaining and preparing experimental flies
<ol>
	<li>Maintain flies in vials containing 10 mL of freshly made food (1% agar, 2.4% brewer&#8217;s yeast, 3% sucrose, 5% cornmeal) in an incubator at 25 &#176;C with 60% humidity. Set the light cycle of the incubator to 12-h light:12-h dark.</li>
	<li>To ensure that a large number of the desired genotype flies ecloses simultaneously, culture young flies (1&#8722;3 days old) in standard food with dry yeast on the surface for 3 days. Transfer the adults to a new food vial with standard food including wet yeast, for 2 days to allow egg laying. Leave the eggs in the incubator to develop and transfer adult flies to a new vial to collect more eggs.</li>
	<li>Collect the eclosed male or female flies each day and culture them in new vials with standard food at the maintenance condition to the desired age. 
	NOTE: To get more same-age flies, multiple vials of the desired genotype may be set up simultaneously. The vials for adult fly culture should be changed every 3&#8722;5 days.</li>
</ol>
2. Counting crop contractions
<ol>
	<li>Anesthetize the flies with CO2 and take one fly into a dissecting plate well containing 200 &#956;L of 1x phosphate buffered saline (PBS, pH = 7.4) composed of 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4, and 1.76 mM KH2PO4.</li>
	<li>Grasp the fly at its thorax using one pair of tweezers, smoothly open the thorax using another pair of tweezers, and then pull the end in opposite directions to open the abdomen. Take the crop and the gut out from the body carefully.</li>
	<li>Wait for the fly to wake up and then visualize the crop and count the number of times it contracts in 1 min. 
	NOTE: Only a complete wave on the crop lobes is counted as one contraction.</li>
	<li>Repeat step 2.3 for 5x between 30 s intervals.</li>
	<li>Calculate the average number of crop contractions per minute. 
	NOTE: During the contraction counting, the fly should be alive, and the gut should be intact and attached at its anterior and posterior ends after dissection.</li>
</ol>
3. Preparing dyed food
<ol>
	<li>Weigh and dissolve the blue dye (Table of Materials) in PBS at a concentration of 20% (w/v).</li>
	<li>Add the 20% blue dye into the boiled liquid maintenance food (step 1.1) with a 1:40 dilution to a final concentration of 0.5% (w/v) during the food cooling process. 
	NOTE: The blue dye is added before the food cooling down and mixed well with stirring. It is optional to dissolve the blue dye in PBS; distilled water is also suitable.</li>
</ol>
4. Feeding flies with dyed food
<ol>
	<li>Transfer groups of same-aged flies to the vials with starvation food (1% agar in distilled water) for 4 h to ensure food intake.</li>
	<li>Transfer the flies to new vials with food dyed blue and culture the flies for the desired time. 
	NOTE: The feeding time is a critical factor and depends on the research purpose. Short feeding, within the time of food passing through, can be used to evaluate the speed of food motility from crop to gut. At the maintenance conditions, the food passes through in about 2 h. However, the time of passing through might be related to culture conditions. Long feeding, up to a few days, can be used to evaluate persistent food distribution status between crop and gut.</li>
</ol>
5. Dissecting flies and collecting dye samples in crop and gut
<ol>
	<li>Anesthetize the flies with CO2 and take one fly into a dissecting plate well containing 200 &#956;L of 1x PBS.</li>
	<li>Grasp the fly at its thorax using one pair of tweezers and take the head off the body using another pair of tweezers. Move the remaining body to a new well containing 200 &#956;L of 1x PBS.</li>
	<li>Wash the body 2x by gently shaking it in 200 &#956;L of 1x PBS using a pair of tweezers to clean the dye attached to the fly body.</li>
	<li>Gently and smoothly open the abdomen using two pairs of tweezers and carefully separate the whole gut from the body.</li>
	<li>Carefully take off the crop from the whole gut and put it in a tube with 100 &#181;L of 1x PBS.</li>
	<li>Lastly, put the whole gut without crop (hereafter referred to as gut) in another tube with 100 &#181;L of 1x PBS.</li>
	<li>Grind the crop and gut respectively in tubes using pipette tips to make the dye dissolve in the PBS.</li>
	<li>Repeat steps 5.1&#8722;5.7 until enough crops and guts are collected for the experiment designed. 
	NOTE: The crop and gut should be fully homogenized, and all dye should be dissolved in the buffer. For research purposes, one or multiple crops or guts can be collected in one tube.</li>
</ol>
6. Calculating dye amounts in crop and gut
<ol>
	<li>Centrifuge the sample tubes at the highest speed for 1 min and transfer 90 &#181;L of supernatant to the wells of a 96 well plate.</li>
	<li>Make a series of blue dye dilutions at concentrations from 1 x 10-7 g/mL to 1 x 10-4 g/mL as standards.</li>
	<li>Add a series of 90 &#181;L standards to the wells of the 96 well plate.</li>
	<li>Measure the absorbance of the samples and standards at 630 nm with a plate spectrophotometer.</li>
	<li>To create a standard curve, plot a line graph of absorbance vs. concentration for each of the standards. Then draw a line of best fit through the points to get the equation used to calculate the dye concentration in the samples.</li>
	<li>Calculate the amount of dye by multiplying the sample concentration by 0.1 mL.</li>
</ol>

<ol>
	<li>Kusano, M., 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=Gastrointestinal+motility+and+functional+gastrointestinal+diseases.">Gastrointestinal motility and functional gastrointestinal diseases.</a> Current Pharmaceutical Design. 20 (16), 2775-2782 (2014).</li><li>Reiter, L. T., Potocki, L., Chien, S., Gribskov, M., Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+analysis+of+human+disease-associated+gene+sequences+in+Drosophila+melanogaster.">A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster.</a> Genome Research. 11 (6), 1114-1125 (2001).</li><li>Apidianakis, Y., Rahme, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+as+a+model+for+human+intestinal+infection+and+pathology.">Drosophila melanogaster as a model for human intestinal infection and pathology.</a> Disease Models &amp; Mechanisms. 4 (1), 21-30 (2011).</li><li>Lemaitre, B., Miguel-Aliaga, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Digestive+Tract+of+Drosophila+melanogaster.">The Digestive Tract of Drosophila melanogaster.</a> Annual Review of Genetics. 47, 377-404 (2013).</li><li>Miguel-Aliaga, I., Jasper, H., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+Physiology+of+the+Digestive+Tract+of+Drosophila+melanogaster.">Anatomy and Physiology of the Digestive Tract of Drosophila melanogaster.</a> Genetics. 210 (2), 357-396 (2018).</li><li>Strand, M., Micchelli, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quiescent+gastric+stem+cells+maintain+the+adult+Drosophila+stomach.">Quiescent gastric stem cells maintain the adult Drosophila stomach.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (43), 17696-17701 (2011).</li><li>Ren, J., 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=Beadex+affects+gastric+emptying+in+Drosophila.">Beadex affects gastric emptying in Drosophila.</a> Cell Research. 24 (5), 636-639 (2014).</li><li>Xi, J., 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=The+TORC1+inhibitor+Nprl2+protects+age-related+digestive+function+in+Drosophila.">The TORC1 inhibitor Nprl2 protects age-related digestive function in Drosophila.</a> Aging. 11 (21), 9811-9828 (2019).</li><li>Wei, Y., Reveal, B., Cai, W., Lilly, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GATOR1+Complex+Regulates+Metabolic+Homeostasis+and+the+Response+to+Nutrient+Stress+in+Drosophila+melanogaster.">The GATOR1 Complex Regulates Metabolic Homeostasis and the Response to Nutrient Stress in Drosophila melanogaster.</a> G3. 6 (12), 3859-3867 (2016).</li><li>Ja, W. W., 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=Prandiology+of+Drosophila+and+the+CAFE+assay.">Prandiology of Drosophila and the CAFE assay.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (20), 8253-8256 (2007).</li><li>Diegelmann, S., 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=The+CApillary+FEeder+Assay+Measures+Food+Intake+in+Drosophila+melanogaster.">The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster.</a> Journal of Visualized Experiments. (121), e55024 (2017).</li><li>Edgecomb, R. S., Harth, C. E., Schneiderman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+feeding+behavior+in+adult+Drosophila+melanogaster+varies+with+feeding+regime+and+nutritional+state.">Regulation of feeding behavior in adult Drosophila melanogaster varies with feeding regime and nutritional state.</a> Journal of Experimental Biology. 197, 215-235 (1994).</li><li>Peller, C. R., Bacon, E. M., Bucheger, J. A., Blumenthal, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+gut+function+in+drop-dead+mutant+Drosophila.">Defective gut function in drop-dead mutant Drosophila.</a> Journal of Insect Physiology. 55 (9), 834-839 (2009).</li><li>Chtarbanova, S., 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=Drosophila+C+virus+systemic+infection+leads+to+intestinal+obstruction.">Drosophila C virus systemic infection leads to intestinal obstruction.</a> Journal of Virology. 88 (24), 14057-14069 (2014).</li><li>Solari, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposite+effects+of+5-HT/AKH+and+octopamine+on+the+crop+contractions+in+adult+Drosophila+melanogaster:+Evidence+of+a+double+brain-gut+serotonergic+circuitry.">Opposite effects of 5-HT/AKH and octopamine on the crop contractions in adult Drosophila melanogaster: Evidence of a double brain-gut serotonergic circuitry.</a> PLoS One. 12 (3), 0174172 (2017).</li></ol>

The authors have nothing to disclose.

In Drosophila ingested food moves from the crop to the gut for digestion. During this process, the nutrients are absorbed, and the waste is expelled out of the body as feces. Thus, comparing food ingestion together with feces ejection can be used to roughly assess the speed of food movement in the body. The method of capillary feeder (CAFE) is widely used to measure food ingestion10,11. The method of feces number counting can be used to estimate the amou...

Most animals have a digestive tube called the gastrointestinal (GI) tract to absorb energy and nutrients from the environment. The human GI tract is composed of four parts: the esophagus, stomach, small intestine, and large intestine (colon). Food passage from the stomach to the intestine is essential for nutrient absorption. Some effectors, such as aging, toxic drugs, and infection, cause disordered GI tract motility and gastric emptying, which is related to some diseases and their symptoms such as dyspepsia, gastroesophageal reflux disease, and constipation1.
The fruit fly (Drosophila melanogaster) is a widely used model animal in biomedical research due to its easy genetic manipulation. Importantly, about 77% of genes associated with human disease have a homolog in Drosophila2. Research using Drosophila has made enormous advances in our understanding of many disease mechanisms. As a powerful genetic model organism, Drosophila is widely used in GI tract research3. Drosophila has a simpler digestive tract, which is divided into three discrete domains: foregut, midgut, and hindgut4. The crop, a part of the foregut, is a bag-like structure that serves as a site for ingested food storage. The midgut is a long tube and functions as the site for food digestion and nutrient absorption through the epithelial layer, which consists of absorptive enterocytes (ECs) and secretory enteroendocrine (EE) cells5. Interestingly, the stomach function in Drosophila is divided into two parts: the crop functions as food storage and the copper cell region (CCR) is a highly acidic region with a pH &#60; 36. In Drosophila, the ingested food is initially moved to the crop and subsequently pumped into the midgut7. Thus, the crop plays a critical role in food passaging. Enveloped by visceral muscles and consisting of a complex array of valves and sphincters, the crop keeps contracting and moving food into the midgut for further digestion.
This protocol allows for the detection of food movement from the crop to the midgut in Drosophila. Crop contraction is evaluated by counting crop contraction frequency. In addition, the effect of the crop on food passaging is investigated by detecting the food distribution between crop and gut. Furthermore, the food distribution can be used to reflect immediate food movement or basic food status using different feeding periods. Taken together, this protocol provides methods to rapidly evaluate crop motility and food passaging in Drosophila.

<table>
	<tbody>
		<tr>
			<td>96-well plate</td>
			<td>Thermo fisher</td>
			<td>269620</td>
			<td></td>
		</tr>
		<tr>
			<td>Brillant Blue FCF</td>
			<td>Solarbio</td>
			<td>E8500</td>
			<td>also called FD&#38;C Blue No. 1</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo fisher</td>
			<td>Heraeus Pico 17</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Spectra Max</td>
			<td>cMax plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Dumont</td>
			<td>11252-30</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring crop motility and food passaging in drosophila

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. Disordered GI motility and gastric emptying cause multiple diseases and symptoms. As a powerful genetic model organism, Drosophila can be used in GI motility research. The Drosophila crop is an organ that contracts and moves food into the midgut for further digestion, functionally similar to a mammalian stomach. Presented is a protocol to study Drosophila crop motility using simple measurement tools. A method for counting crop contractions to evaluate crop motility and a method for detecting the distribution of food dyed blue between the crop and gut using a spectrophotometer to investigate the effect of the crop on food passaging is described. The method was used to detect the difference in crop motility between control and nprl2 mutant flies. This protocol is both cost-efficient and highly sensitive to crop motility.

These methods to count crop contraction rate and detect dyed food distribution can be used to evaluate crop function on food motility. The crop contraction reflects the frequency of pushing food into the gut. The distribution of dye in the fly after a short feeding period indicates immediate food passaging from crop to midgut.
Target of rapamycin complex 1 (TORC1) is a master regulator that mediates nutrient and cell metabolism. TORC1 inhibition extends lifespan in many organisms, including <e...

Watch this Scientific Journal Video about Measuring Crop Motility and Food Passaging in Drosophila at JoVE.com

Measuring Crop Motility and Food Passaging in Drosophila

The goal of this protocol is to measure crop contraction and quantify food distribution in the Drosophila gut.

Measuring Crop Motility and Food Passaging in Drosophila

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. ...

measuring-crop-motility-and-food-passaging-in-drosophila

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Biology

5.4K Views.  Yangzhou University. The goal of this protocol is to measure crop contraction and quantify food distribution in the Drosophila gut.

Video: Measuring Crop Motility and Food Passaging in Drosophila

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Catheterization of Intestinal Loops in Ruminants

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the outside world. As such, the intestine plays a pivotal role in immunosurveillance and protection from enteric pathogens. Investigating intestinal physiology and immunology commonly employs 'intestinal loops' as an experimental model. The majority of these loop models are non-recovery surgical procedures that study short-term (&lt;24 hr) changes in the intestine (1-3). We previously created a recovery intestinal loop model to specifically measure long-term (&lt;6 mo) immunological changes in the intestine of sheep following exposure to vaccines, adjuvants, and viruses (4). This procedure localized treatments to a specific 'loop', allowing us to sample this area of the intestine. A significant drawback of this method is the single window of opportunity to administer treatments (i.e. at the time of surgery). Furthermore, samples of both the intestinal mucosa and luminal contents can only be taken at the termination of the project. Other salient limitations of the above model are that the surgical manipulation and requisite post-operative measures (e.g. administration of antibiotics and analgesics) can directly affect the treatment itself and/or alter immune function, thereby confounding results. Therefore, we modified our intestinal loop model by inserting long-term catheters into the loops. Sheep recover fully from the procedure, and are unaffected by the exteriorized catheters. Notably, the establishment of catheters in loops allows us to introduce multiple treatments over an extended interval, following recovery from surgery and clearance of drugs administered during surgery and the post-operative period.

We describe a novel surgical method for catheterizing 'intestinal loops' within the ileum of sheep. Once animals have recovered from surgery and have cleared antibiotics and analgesics, multiple treatments can be deposited directly in loops via the catheters.

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the ...

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Gastrointestinal Motility Monitor (GIMM)

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility in isolated segments of guinea pig distal colon. The complete system consists of a computer, video camera, illuminated organ bath, peristaltic and heated water bath circulating pumps, and custom GIMM software to record and analyze data. Compared with traditional methods of monitoring colonic peristalsis, the GIMM system allows for continuous, quantitative evaluation of motility. The guinea pig distal colon is bathed in warmed, oxygenated Krebs solution, and fecal pellets inserted in the oral end are propelled along the segment of colon at a rate of about 2 mm/sec. Movies of the fecal pellet proceeding along the segment are captured, and the GIMM software can be used track the progress of the fecal pellet. Rates of propulsive motility can be obtained for the entire segment or for any particular region of interest. In addition to analysis of bolus-induced motility patterns, spatiotemporal maps can be constructed from captured video segments to assess spontaneous motor activity patterns. Applications of this system include pharmacological evaluation of the effects of receptor agonists and antagonists on propulsive motility, as well as assessment of changes that result from pathophysiological conditions, such as inflammation or stress. The guinea pig distal colon propulsive motility assay, using the GIMM system, is straightforward and simple to learn, and it provides a reliable and reproducible method of assessing propulsive motility.

Gastrointestinal Motility Monitor GIMM

Evaluation of colonic motility in the guinea pig distal colon with the Gastrointestinal Motility Monitor (GIMM) is a straightforward and simple to learn approach to quantitatively evaluate propulsive motility in the gastrointestinal tract.

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...