We thank B. Spee for generously providing LD540. This work was supported by a Netherlands Organization for Scientific Research grant (NWO-ZonMW; VIDI 016.146.353) to S.M.

All experimentation using human tissues described herein was approved by the ethical committee at University Medical Center Utrecht (UMCU). Informed consent for tissue collection, generation, storage, and use of the organoids was obtained from patients at the Wilhelmina Children&#39;s Hospital (WKZ)-UMCU.
1. Preparation of Culture Media
NOTE: This protocol should be performed inside a biosafety cabinet. The organoids should be handled according to standard cell culture guidelines.
<ol>
	<li>Prepare basal culture medium. 
	NOTE: Culture medium without growth factors is referred to as basal medium (BM).
	<ol>
		<li>Add 5 mL of HEPES (1 M), 5 mL of L-glutamin (100x) and 5 mL of penicillin-streptomycin (5,000 U/mL) to 500 mL of advanced Dulbecco&#39;s modified Eagle medium with Ham&#39;s nutrient mixture F-12 to prepare BM medium.</li>
		<li>Store the prepared BM medium at 4 &#176;C and use for a maximum of 2 months.</li>
	</ol>
	</li>
	<li>Prepare R-spondin and Noggin conditioned medium (CM) according the manufacturer&#39;s protocol.
	<ol>
		<li>Briefly, grow up the cells to the desired quantity in hyperflasks with 5 x 107&#160;cells in 555 mL medium without selective antibiotic per flask.</li>
		<li>Grow the cells for 4 days until confluent.</li>
		<li>Replace the medium with BM and culture for 8 additional days.</li>
		<li>After 8 days of culture at 37 &#176;C, collect the culture medium and centrifuge for 5 min at 450 x g to pellet any remaining cells.</li>
		<li>Filter sterilize the supernatant, and store aliquots of the R-spondin or Noggin CM at -20 &#176;C for a maximum of 6 months.</li>
	</ol>
	</li>
	<li>Prepare Wnt3A-CM according to Boj et al.15.
	<ol>
		<li>Briefly, grow up the cells to the desired quantity in 145 mm dishes with 2 x 106 cells in 20 mL medium without selective antibiotic per dish. Wrap each dish with plastic foil to avoid evaporation of the culture medium.</li>
		<li>After 8 days of culture at 37 &#176;C, collect the culture medium and centrifuge for 5 min at 450 x g to pellet any remaining cells.</li>
		<li>Filter sterilize the supernatant, and store aliquots of the Wnt3A-CM at 4 &#176;C for a maximum of 2 months.</li>
	</ol>
	</li>
	<li>Prepare organoid expansion medium. 
	NOTE: Human small intestinal organoid expansion medium (see recipe in Supplemental Table 1) is referred to as hSI-EM. The following steps will produce a final volume of 1 L hSI-EM, and can be scaled up or down as required.
	<ol>
		<li>Dissolve 1.46 g of nicotinamide in 12 mL of cell culture grade phosphate-buffered saline (PBS) to create a final dilution of 1 M.</li>
		<li>Dissolve 245 mg of n-acetyl cysteine in 3 mL of cell culture grade PBS to create a final dilution of 500 mM. 
		NOTE: To accelerate the formation of a solution, the nicotinamide and n-acetyl cysteine can be incubated in a water bath at 37 &#176;C. Both solutions can be prepared in batch, aliquoted, and stored at -20 &#176;C for future use.</li>
		<li>Filter sterilize both solutions through a 0.22 &#181;m filter into a sterile 15 mL tube.</li>
		<li>Add 167 mL of BM to a sterile 500 mL culture medium flask. Add 200 mL of R-Spondin-CM, 100 mL of noggin-CM, and 100 &#181;L of recombinant mEGF (500 &#181;g/mL) to a final concentration of 50 ng/mL.</li>
		<li>Add 10 mL of the sterilized nicotinamide solution and 2.5 mL of the sterilized n-acetyl cysteine solution. Add 20 mL of B27 supplement (50x). 
		NOTE: At this stage, the medium is referred to as hSI-EM without WAS (Wnt3A-CM, A83-01, and SB202190), and can be aliquoted and stored at -20 &#176;C. If the medium is aliquoted into smaller volumes, adjust the concentrations of the following steps accordingly.</li>
		<li>To 500 mL of hSI-EM without WAS, add 10 mL of Wnt3A-CM of the last freshly prepared batch and 10 mL of Wnt3A-CM of the second last batch to minimize variability changes in Wnt3A-CM conditions.</li>
		<li>Add A83-01 to a final concentration of 500 nM, and SB202190 to a final concentration of 10 &#181;M.</li>
		<li>Store the prepared hSI-EM (with WAS) at 4 &#176;C and use for a maximum of 2 weeks. 
		NOTE: Add additional Y-27632 to a final concentration of 10 &#181;M (referred to as hSI-EM+Y) when crypts or single cells are cultured or organoids are started from cryopreservation.</li>
	</ol>
	</li>
	<li>Prepare fluorescent activated cell sorting (FACS) buffer.
	<ol>
		<li>Add 10 mL of fetal calf serum (FCS) to 40 mL of PBS without Ca2+/Mg2+ for a final concentration of 10% FCS. 
		NOTE: The FCS prevents cells from adhering to labware.</li>
	</ol>
	</li>
</ol>
2. Culture Procedures for Human Small Intestinal Organoids
NOTE: This protocol should be performed inside a biosafety cabinet. The organoids should be handled according to standard cell culture guidelines. When handling organoids or organoid-derived cells, the cells should be kept on ice whenever possible. The cells will remain viable for a few hours after harvesting when this is ensured. Organoids should be cultured in a standard cell culture incubator at 37 &#176;C with 5% CO2. These conditions apply to all incubation steps with organoids embedded in basement membrane matrix (BMM; i.e., Matrigel) throughout this protocol. The authors have used duodenum-derived organoids for these assays.
<ol>
	<li>Passaging organoids 
	NOTE: Every organoid culture has its own doubling time. Normally, small intestinal organoids can be passaged 1:3&#8722;1:5 every 7&#8722;10 days. When passaged as single cells, passaging efficiency can be up to 1:20, depending on cell density. For establishment and maintenance, organoids are cultured in 24-well plates; for the LDF assay, in either 24- or 96-well plates.
	<ol>
		<li>Preparation
		<ol>
			<li>Pre-warm unpacked 24-well tissue culture plates in an incubator at 37 &#176;C, 5 % CO2 at least overnight and preferably 5 days in advance. 
			NOTE: Pre-warming the tissue culture plates in the cell culture incubator ensures proper formation and attachment of organoid-containing droplets of BMM.</li>
			<li>To reduce the number of freeze-thaw cycles, prepare 1 mL aliquots of BMM and store at -20 &#176;C. Thaw a vial of BMM on ice at least 30 min before starting the organoid passaging procedure.</li>
			<li>Keep a 50 mL tube of BM on ice as washing medium.</li>
			<li>Prepare hSI-EM as described in section 1.4, and prepare an appropriate amount of hSI-EM+Y, for example, 15 mL for a full 24-well plate.</li>
		</ol>
		</li>
		<li>Collect organoids.
		<ol>
			<li>Carefully aspirate the culture medium without disturbing the droplets of BMM.</li>
			<li>Add 500 &#181;L of cold BM to the first well of organoids, and disrupt the BMM droplets with organoids by pipetting gently up and down with a P1000 pipet.</li>
			<li>Repeat this procedure with the same medium in the next well as required, but do not harvest more than 2 wells per 500 &#181;L of BM.</li>
			<li>Collect the organoids in a low-binding 1.5 mL microcentrifuge tube and spin down in a mini tabletop centrifuge for 15&#8722;20 s (max. 2,000 x g). Aspirate the supernatant completely and remove the last bit of medium with a P200 pipet. 
			NOTE: When the organoids cultures look clean under a microscope and do not contain any dead cells or other debris, the BMM drops can be harvested directly in trypsin instead of BM in step 2.1.2.2. After step 2.1.2.3, proceed directly to incubation in step 2.1.3.1.</li>
		</ol>
		</li>
		<li>Dissociate the organoids into single cells.
		<ol>
			<li>Add 400 &#181;L of trypsin to the spun down organoids. Incubate the organoids at 37 &#176;C for 5 min in a water bath.</li>
			<li>Disrupt the remaining aggregates of cells by pipetting up and down gently with a P200 pipet. Again, incubate the organoids at 37 &#176;C for 5 min in a water bath.</li>
			<li>Check the progress of cell dissociation under a microscope using 4x magnification. Repeat manual disruption and incubation when large clumps of cells remain.</li>
			<li>When only single cells remain, add 1 mL of BM and spin down the cells in a mini tabletop centrifuge for 15&#8722;20 s.</li>
			<li>Aspirate the supernatant completely. Resuspend the single cells in 200 &#181;L of fresh hSI-EM using a P200 pipet. Add an additional 800 &#181;L of hSI-EM.</li>
			<li>Count the number of cells in suspension. 
			NOTE: In the authors&#39; lab, manual counting of dissociated organoid cells yields more reliable results than an automated cell counter. When an automated cell counter is used, the cell density for downstream applications should be tested in-house and adjusted if too little or too many organoids grow out.</li>
		</ol>
		</li>
		<li>Seed organoids for maintenance or lipid droplet formation assay.
		<ol>
			<li>Calculate the volume of cells which is needed for the final cell density. 
			NOTE: Approximately 250 cells/&#181;L is an appropriate density. In a 24-well plate, 30 &#181;L is seeded into each well, while 5 &#181;L is seeded into each well of a 96-well plate.</li>
			<li>Take out the appropriate volume of suspension and adjust the density to 750 cells/&#181;L (or spin down and resuspend when the density is too low).</li>
			<li>Add BMM to the cell suspension in a ratio of 2:1. The final cell density in this mixture is 250 cells/&#181;L. Gently mix the suspension by pipetting, carefully avoiding any bubbles.</li>
			<li>In a pre-warmed tissue culture plate, seed out three 10 &#181;L droplets per well in a 24-well plate or a single 5 &#181;L droplet per well in a 96-well plate.</li>
			<li>Place the plate in an incubator at 37 &#176;C, 5% CO2 for 10&#8722;15 min to solidify the BMM droplets. Meanwhile, pre-warm an appropriate amount of hSI-EM+Y in a water bath at 37 &#176;C.</li>
			<li>Carefully add 500 &#181;L of pre-warmed hSI-EM+Y to each well of a 24-well plate or 100 &#181;L to each well of a 96-well plate. Incubate the cells in an incubator at 37 &#176;C, 5% CO2 and after 2&#8722;3 days change the medium to hSI-EM (without Y) and refresh 2&#8722;3 times a week. 
			NOTE: After 7&#8722;10 days, a maintenance culture of organoids should be passaged again.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Lipid Droplet Formation Assay
<ol>
	<li>Preparation of oleic acid conjugate 
	NOTE: Since oleic acid (OA) is hydrophobic and not soluble in water, it is conjugated to bovine serum albumin (BSA). BSA can bind multiple fatty acid molecules and is in this case used in a 1:8 ratio to make oleic acid accessible to the intestinal cells. Free fatty acids and BSA can both bind to plastic labware. To ensure a proper final concentration, use glass vials and glass pipets whenever possible.
	<ol>
		<li>Weigh 0.2 g of liquid oleic acid at room temperature. Add 1.5 mL of culture-grade sterile PBS and heat the mixture to 70 &#176;C for 1 h. Vortex intermittently.</li>
		<li>Weigh 5.89 g of fatty acid-free BSA and dissolve in 33.9 mL of PBS. Warm the mixture in a water bath at 37 &#176;C until the BSA is fully dissolved.</li>
		<li>Vortex the OA mixture again to create an emulsion of fine droplets and immediately add it to the BSA solution using a glass pipet. Keep the final solution close to 37 &#176;C after adding the OA. The final mixture consists of 20 mM OA in 2.5 mM BSA.</li>
		<li>Keep the mixture at 37 &#176;C for 30 min until a clear yellowish solution remains.</li>
		<li>The OA-BSA conjugate can be aliquoted and frozen at -20 &#176;C for at least 6 months. Upon thawing an aliquot, incubate it at 37 &#176;C until the cloudiness dissolves and the mixture is clear again. 
		NOTE: Because of the high protein and lipid content of the final solution, the mixture cannot be filter sterilized or autoclaved. When the components were handled with care, in a laminar flow hood when possible and using sterile PBS, the authors did not experience microbial infections.</li>
	</ol>
	</li>
	<li>LDF confocal assay
	<ol>
		<li>Sample preparation
		<ol>
			<li>Passage organoids as described in section 2.1. Seed out the organoid-derived single cells in a black clear-bottom 96-well plate.</li>
			<li>On day 6 of culture on hSI-EM, replace the culture medium with hSI-EM containing 1 mM OA-BSA conjugate.</li>
			<li>Incubate the cells for 16&#8722;17 h (overnight) at 37 &#176;C in presence or absence of 0.1 &#181;M DGAT1 inhibitor. Include a vehicle control of 2.5 mM BSA.</li>
			<li>After 16&#8722;17 h, aspirate the medium without disturbing the BMM droplets. Fixate the organoids by adding 100 &#181;L of 4% neutral buffered formaldehyde to the wells for 30 min at room temperature. 
			NOTE: The formaldehyde will partially dissolve the BMM, while the organoids sink to the bottom and adhere to the bottom of the plate.</li>
			<li>Remove the formaldehyde gently, and carefully wash the wells with 150 &#181;L of PBS per well.</li>
			<li>Stain the cells for LDs with 0.025 mg/mL LD540 and 4&#8242;,6-diamidino-2-phenylindole (DAPI) in PBS for 15 min at room temperature in the dark.</li>
			<li>Wash the wells carefully with PBS. 
			NOTE: The protocol can be paused here when necessary. Keep the samples covered in PBS in the dark at 4 &#176;C. The LD540 staining will remain stable for up to a week. This assay can also be performed with live cells, to monitor LD formation over a period of time. For this, the fixation has to be omitted from the protocol, and the DAPI should be replaced with Hoechst staining. LD540 can stain LDs in living cells in the same concentration as described.</li>
		</ol>
		</li>
		<li>Confocal imaging 
		NOTE: The imaging can be performed on prepared organoid samples in the black clear-bottom 96-well plate.
		<ol>
			<li>For overview images of whole organoids, use a 40x objective suited for confocal fluorescent imaging.</li>
			<li>Set the microscope to image the DAPI channel at 405 nm excitation and ca. 410&#8722;535 nm emission wavelength. For the LD540 dye choose an excitation laser at 540 nm (543 nm is optimal) and set the emission filters to 545&#8722;700 nm. 
			NOTE: In this protocol, a laser-scanning confocal system with a white light laser, acousto-optical beam splitter (AOBS), 10x/20x objective, and spectral detection system was used. This allows for exact tuning to the specific wavelengths. If a comparable system is not available, choose laser lines and short-/long-pass filters close to the specifications above. For whole-organoid imaging, a resolution of 512 x 512 or 1024 x 1024 is sufficient for downstream image analysis.</li>
			<li>Set the pinhole size to 1 airy unit (AU) for sufficient z-axis resolution.</li>
			<li>To image one half of a spherical organoid, set a z-stack to approximately 85 &#181;m.</li>
		</ol>
		</li>
		<li>Image analysis 
		NOTE: For image analysis, Fiji/ImageJ16,17 was used to generate maximum projections. The analysis can be performed with any image analysis software package which allows for maximum projection, manual thresholding, and particle analysis.
		<ol>
			<li>Using Fiji/ImageJ, transform the z-stack of each organoid to a maximum projection: Image | Stacks | Z-project.</li>
			<li>Set the threshold of the maximum projection to a level in which there is no LD540 signal visible in the BSA vehicle control sample: Image | Adjust | Threshold. Use these settings to threshold each image.</li>
			<li>Measure the total area of fluorescence for each maximum projection using the function Analyze | Analyze particles. 
			NOTE: Although the assay resolution is better using a confocal microscope, this assay can also be analyzed by using a fluorescent plate reader with similar filters. To normalize for the organoid count per well, the LD540 signal should be divided by the DAPI signal. Finally, the signal of the BSA vehicle control should be subtracted to normalize the measurements. This approach allows the assay to be scaled towards a 96-well plate format.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>LDF flow cytometric assay
	<ol>
		<li>Sample preparation
		<ol>
			<li>Passage organoids as described in section 2.1. Seed out the organoid-derived single cells in a 24-well tissue culture plate. Two wells per condition is sufficient for flow cytometry.</li>
			<li>On day 10 of culture on hSI-EM, replace the culture medium with hSI-EM containing 1 mM OA-BSA conjugate. 
			NOTE: In contrast with the confocal analysis, 10 days of growth in EM was chosen for the flow cytometry assay instead of 6 days. The extra 4 days of expansion will result in optically overlapping organoids which would complicate the microscopy-based assay. However, the greater number of cells facilitates flow cytometry analysis.</li>
			<li>Incubate the cells for 16&#8722;17 h (overnight) at 37 &#176;C in presence or absence of 0.1 &#181;M DGAT1 inhibitor. Include a vehicle control of 2.5 mM BSA.</li>
			<li>After 16&#8722;17 h, collect the organoids as described in section 2.1.2.</li>
			<li>Dissociate the organoids into single cells as described in section 2.1.3. 
			NOTE: Although not strictly required, it is recommended to check the cell count in each sample. A total count of 10,000 cells per sample is the absolute minimum required for sufficient resolution. For optimal results use ca. 50,000&#8722;100,000 cells.</li>
			<li>Spin down the cells in a mini tabletop centrifuge for 15&#8722;20 s.</li>
			<li>Stain each sample with 500 &#181;L of 0.025 mg/mL LD540 and 1 &#181;g/mL Hoechst in PBS for 15 min at room temperature in the dark.</li>
			<li>Spin down the cells in a mini tabletop centrifuge for 15&#8722;20 s and wash 3x with PBS.</li>
			<li>Fixate the cells by resuspending them in 500 &#181;L of 4% neutral buffered formaldehyde for 15 min at room temperature.</li>
			<li>Spin down the cells and wash 3x with FACS buffer.</li>
			<li>Pre-rinse FACS tubes with FACS buffer to prevent cells sticking to the tube wall.</li>
			<li>Resuspend the cells in 200 &#181;L of FACS buffer and transfer the cell suspension to the pre-rinsed FACS tubes.</li>
		</ol>
		</li>
		<li>Flow cytometric analysis
		<ol>
			<li>Set the gating parameters to exclude dead cells and clumps of cells (see the representative results section).</li>
			<li>In the final gated population, measure at least 10,000 cells for reliable results. 
			NOTE: From this population, the mean fluorescent intensity (MFI) of LD540 and the mean SSC-A signal together provide a measure of the total volume of LD formation per cell.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Yen, C. L. E., Nelson, D. W., Yen, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+triacylglycerol+synthesis+in+fat+absorption+and+systemic+energy+metabolism.">Intestinal triacylglycerol synthesis in fat absorption and systemic energy metabolism.</a> Journal of Lipid Research. 56 (3), 489-501 (2015).</li><li>Yen, C. L. E., Stone, S. J., Koliwad, S., Harris, C., Farese, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thematic+review+series:+glycerolipids.+DGAT+enzymes+and+triacylglycerol+biosynthesis.">Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis.</a> Journal of Lipid Research. 49 (11), 2283-2301 (2008).</li><li>D'Aquila, T., Hung, Y. H., Carreiro, A., Buhman, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+discoveries+on+absorption+of+dietary+fat:+Presence,+synthesis,+and+metabolism+of+cytoplasmic+lipid+droplets+within+enterocytes.">Recent discoveries on absorption of dietary fat: Presence, synthesis, and metabolism of cytoplasmic lipid droplets within enterocytes.</a> Biochimica et Biophysica Acta. 1861 (8 Pt A), 730-747 (2016).</li><li>Chavez-Jauregui, R. N., Mattes, R. D., Parks, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+fat+absorption+and+effect+of+sham+feeding+on+postprandial+lipema.">Dynamics of fat absorption and effect of sham feeding on postprandial lipema.</a> Gastroenterology. 139 (5), 1538-1548 (2010).</li><li>Chitraju, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triglyceride+Synthesis+by+DGAT1+Protects+Adipocytes+from+Lipid-Induced+ER+Stress+during+Lipolysis.">Triglyceride Synthesis by DGAT1 Protects Adipocytes from Lipid-Induced ER Stress during Lipolysis.</a> Cell Metabolism. 26 (2), 407-418 (2017).</li><li>van Rijn, J. 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=Intestinal+failure+and+aberrant+lipid+metabolism+in+patients+with+DGAT1+deficiency.">Intestinal failure and aberrant lipid metabolism in patients with DGAT1 deficiency.</a> Gastroenterology. 1, 130-143 (2018).</li><li>Haas, J. T., 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=DGAT1+mutation+is+linked+to+a+congenital+diarrheal+disorder.">DGAT1 mutation is linked to a congenital diarrheal disorder.</a> Journal of Clinical Investigation. 122 (12), 4680-4684 (2012).</li><li>Gluchowski, N. L., 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=Identification+and+characterization+of+a+novel+DGAT1+missense+mutation+associated+with+congenital+diarrhea.">Identification and characterization of a novel DGAT1 missense mutation associated with congenital diarrhea.</a> Journal of Lipid Research. 58 (6), 1230-1237 (2017).</li><li>Ratchford, T. L., Kirby, A. J., Pinz, H., Patel, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congenital+Diarrhea+From+DGAT1+Mutation+Leading+to+Electrolyte+Derangements,+Protein-losing+Enteropathy,+and+Rickets.">Congenital Diarrhea From DGAT1 Mutation Leading to Electrolyte Derangements, Protein-losing Enteropathy, and Rickets.</a> Journal of Pediatric Gastroenterology and Nutrition. 66 (3), e82-e83 (2018).</li><li>Stephen, 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=Congenital+protein+losing+enteropathy:+an+inborn+error+of+lipid+metabolism+due+to+DGAT1+mutations.">Congenital protein losing enteropathy: an inborn error of lipid metabolism due to DGAT1 mutations.</a> European Journal of Human Genetics. 24 (9), 1268-1273 (2016).</li><li>Schlegel, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+deficits+in+apical+transporter+trafficking+associated+with+deficiency+in+diacylglycerol+acyltransferase.">Reversible deficits in apical transporter trafficking associated with deficiency in diacylglycerol acyltransferase.</a> Traffic (Copenhagen, Denmark). 19 (11), 879-892 (2018).</li><li>Wilfling, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triacylglycerol+synthesis+enzymes+mediate+lipid+droplet+growth+by+relocalizing+from+the+ER+to+lipid+droplets.">Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets.</a> Developmental Cell. 24 (4), 384-399 (2013).</li><li>Sato, T., 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=Long-term+expansion+of+epithelial+organoids+from+human+colon,+adenoma,+adenocarcinoma,+and+Barrett's+epithelium.">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.</a> Gastroenterology. 141 (5), 1762-1772 (2011).</li><li>Middendorp, 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=Adult+stem+cells+in+the+small+intestine+are+intrinsically+programmed+with+their+location-specific+function.">Adult stem cells in the small intestine are intrinsically programmed with their location-specific function.</a> Stem Cells. 32 (5), 1083-1091 (2014).</li><li>Boj, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forskolin-induced+Swelling+in+Intestinal+Organoids:+An+In+Vitro+Assay+for+Assessing+Drug+Response+in+Cystic+Fibrosis+Patients.">Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients.</a> Journal of Visualized Experiments. (120), e55159 (2017).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Rueden, C. T., 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=ImageJ2:+ImageJ+for+the+next+generation+of+scientific+image+data.">ImageJ2: ImageJ for the next generation of scientific image data.</a> BMC Bioinformatics. 18 (1), 529 (2017).</li><li>Spandl, J., White, D. J., Peychl, J., Thiele, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+multicolor+imaging+of+lipid+droplets+with+a+new+dye,+LD540.">Live cell multicolor imaging of lipid droplets with a new dye, LD540.</a> Traffic. 10 (11), 1579-1584 (2009).</li><li>Hung, Y. H., Carreiro, A. L., Buhman, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dgat1+and+Dgat2+regulate+enterocyte+triacylglycerol+distribution+and+alter+proteins+associated+with+cytoplasmic+lipid+droplets+in+response+to+dietary+fat.">Dgat1 and Dgat2 regulate enterocyte triacylglycerol distribution and alter proteins associated with cytoplasmic lipid droplets in response to dietary fat.</a> Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. 1862 (6), 600-614 (2017).</li></ol>

The authors have nothing to disclose.

Here, we provide a protocol to determine LD formation in human intestinal organoids upon incubation with oleic acid. This method is based on the LD-specific fluorescent dye LD54018, which allows for characterization and quantification of the total volume of lipid droplets within an organoid culture. The procedures to establish and maintain human intestinal organoid cultures have been published before13, and a visual guide of this protocol is available as well<sup class="xre...

Lipids are a crucial component of the human diet and play an important role in systemic energy storage and metabolism. When ingested, dietary lipids are degraded into free fatty acids (FFAs) and monoglycerides (MGs) by pancreatic lipases. These substrates are then taken up by the enterocytes of the intestinal epithelium, where they are first re-esterified to diglycerides (DG) by monoglyceride acyltransferases (MGAT) enzymes and subsequently to triglycerides (TG) by diacylglycerol acyltransferase 1 (DGAT1)1. Finally, these TGs are integrated into either chylomicrons for export to the lymph system or cytosolic lipid droplets (LDs) for intracellular storage2,3. Although chylomicrons are needed to distribute dietary lipids to other organs, the importance of intracellular fat storage in LDs is not completely clear. However, LDs have been shown to perform a regulatory function in the intestine, as they slowly release lipids into the circulation up to 16 h after a meal4. Furthermore, LDs have been shown to protect against toxic fatty acid concentrations, such as in mouse adipocytes during lipolytic conditions5.
The DGAT1 protein is located on the endoplasmic reticulum (ER) membrane and plays a crucial role in LD formation in the intestinal epithelium. Homozygous mutations in DGAT1 lead to early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure upon fat intake, illustrating the importance of DGAT1 in lipid homeostasis of the human intestinal epithelium6,7,8,9,10. Since the occurrence of DGAT1-deficiency in humans is rare, access to primary patient-derived cells has been scarce. Furthermore, the long-term culture of intestinal epithelial cells has long been restricted to tumor-derived cell lines which represent the normal physiology only to a limited extend. Therefore, DGAT1-mediated LD formation has mostly been studied in fibroblasts or animal-derived cell lines7,10,11,12. As such, it was recently shown that DGAT1-deficient patient-derived fibroblasts accumulate less LDs compared to healthy control cells after stimulation with oleic acid (OA)8.
Previously, protocols were established to culture epithelial stem cells from any gastrointestinal organ in the form of three-dimensional (3D) organoids13. These intestinal organoids can be kept in culture for a long period of time13, and allow the functional study of patient- and intestinal location-specific epithelial characteristics14. They are genetically and phenotypically stable and can be stored, allowing long-term expansion and biobanking13.
We recently demonstrated that LD formation can be readily measured in human intestinal organoids in a LD formation (LDF) assay6. When exposed to OA for 16 h, organoids generate LDs to protect the cells from lipid-induced toxicity. When OA concentrations are too high, the cells die by caspase-mediated apoptosis6. The LDF assay was previously shown to be largely dependent on DGAT1 as indicated by organoids derived from DGAT1-mutant patients and by the use of DGAT1-specific inhibitors6.
For the LDF assay described in detail here, 3D organoids are cultured from intestinal biopsies and are passaged weekly by disruption into single cells that easily form new organoids. For running the LDF assay, ~7,500 organoid-derived single cells are plated in each well of a 24-well plate. Organoids are formed over several days, incubated overnight with 1 mM OA and stained with LD540, a fluorescent cell-permeable LD-specific dye that facilitates imaging. The LD formation is then quantified by confocal microscopy, fluorescent plate reader, or flow cytometry.
By scaling this LD formation assay to a 96-well format, the assay can also be used for high-throughput analysis of LD formation to screen for novel drugs which affect LD formation in human intestinal organoid cultures, or to study (human genetic) disorders that affect LD metabolism.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Gibco</td>
			<td>12634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement&#160;</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Basement membrane matrix (matrigel)</td>
			<td>BD Biosciences</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DGAT1 inhibitor (AZD 3988)</td>
			<td>Tocris Bioscience</td>
			<td>4837/10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fatty acid free BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Klinipath</td>
			<td>4078-9001</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamin (GlutaMAX, 100X)</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>laser scanning confocal microscope</td>
			<td>Leica</td>
			<td>SP8X</td>
			<td></td>
		</tr>
		<tr>
			<td>LD540</td>
			<td></td>
			<td></td>
			<td>kindly provided by Dr. B. Spee, Utrecht University</td>
		</tr>
		<tr>
			<td>mEGF</td>
			<td>Peprotech</td>
			<td>315-09_500ug</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Noggin producing cells (HEK293-mNoggin-Fc cells)</td>
			<td></td>
			<td></td>
			<td>MTA with J. den Hertog, Hubrecht Institute</td>
		</tr>
		<tr>
			<td>Oleic acid</td>
			<td>Sigma-Aldrich</td>
			<td>O1008-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>p38 MAPK inhibitor (p38i) (SB202190)</td>
			<td>Sigma-Aldrich</td>
			<td>S7067-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8662-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS without Ca2+/Mg2+</td>
			<td>Sigma-Aldrich</td>
			<td>D8537-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (5,000 U/ml)</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>R-spondin producing cells (Cultrex HA-R-Spondin1-Fc 293T Cells)</td>
			<td>R&#38;D systems</td>
			<td>3710-001-01</td>
			<td></td>
		</tr>
		<tr>
			<td>TC-treated 24 well plates</td>
			<td>Greiner-One</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr>
			<td>TC-treated black clear-bottom 96 well plates</td>
			<td>Corning Life Sciences</td>
			<td>353219</td>
			<td></td>
		</tr>
		<tr>
			<td>TGFb type I receptor inhibitor (A83-01)&#160;</td>
			<td>Tocris Bioscience</td>
			<td>2939/10</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin (TrypLE Express)</td>
			<td>Life Technologies</td>
			<td>12604021</td>
			<td></td>
		</tr>
		<tr>
			<td>WNT-3A producing cells (L-Wnt-3A cells)</td>
			<td></td>
			<td></td>
			<td>MTA with J. den Hertog, Hubrecht Institute</td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride (Rho kinase inhibitor)</td>
			<td>Abcam</td>
			<td>ab120129-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

a fluorescence-based assay for characterization and quantification of lipid droplet formation in human intestinal organoids

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) molecules, before they are packaged into chylomicrons for transport to the lymph or into cytosolic lipid droplets (LDs) for intracellular storage. A crucial step for the formation of LDs is the catalytic activity of diacylglycerol acyltransferases (DGAT) in the final step of TG synthesis. LDs are important to buffer toxic lipid species and regulate cellular metabolism in different cell types. Since the human intestinal epithelium is regularly confronted with high concentrations of lipids, LD formation is of great importance to regulate homeostasis. Here we describe a simple assay for the characterization and quantification of LD formation (LDF) upon stimulation with the most common unsaturated fatty acid, oleic acid, in human intestinal organoids. The LDF assay is based on the LD-specific fluorescent dye LD540, which allows for quantification of LDs by confocal microscopy, fluorescent plate reader, or flow cytometry. The LDF assay can be used to characterize LD formation in human intestinal epithelial cells, or to study human (genetic) disorders that affect LD metabolism, such as DGAT1 deficiency. Furthermore, this assay can also be used in a high-throughput pipeline to test novel therapeutic compounds, which restore defects in LD formation in intestinal or other types of organoids.

For proper analysis of LD formation, the organoids should not be seeded too densely prior to stimulation with OA and subsequent staining. This is especially of importance for the confocal and plate reader readout, since overlapping organoids might interfere with the fluorescence. An example of proper organoid seeding density (Figure 1A) and a culture with overlapping organoids is shown (Figure 1B...

Watch this Scientific Journal Video about A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids at JoVE.com

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

This protocol describes an assay for the characterization of lipid droplet (LD) formation in human intestinal organoids upon stimulation with fatty acids. We discuss how this assay is used for quantification of LD formation, and how it can be used for high throughput screening for drugs that affect LD formation.

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) ...

a-fluorescence-based-assay-for-characterization-quantification-lipid

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Medicine

Unit 1

Biochemistry of the Cell

SCIENTISTS IN ACTION

8.9K Views.  University Medical Center Utrecht, Utrecht University. This protocol describes an assay for the characterization of lipid droplet (LD) formation in human intestinal organoids upon stimulation with fatty acids. We discuss how this assay is used for quantification of LD formation, and how it can be used for high throughput screening for drugs that affect LD formation.

Video: A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor&#39;s function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca2+ release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12). A key advantage of this method is that it also enables screening of compounds endowed with intrinsic agonistic properties (i.e., compounds eliciting an increase in intracellular Ca2+ concentration in the absence of CXCL12) or compounds modulating the receptor&#39;s function via interaction with allosteric binding sites (i.e., positive and negative allosteric modulators (PAMs and NAMs, respectively)). On the down side, autofluorescent compounds might interfere with the assay&#39;s readout, thereby hampering reliable data interpretation. Most likely this assay can be implemented, with minimal adaptations, as a generic drug discovery assay for many other GPCRs of which the activation leads to a release of intracellular Ca2+.

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

The described cellular assay is designed for the identification of CXC chemokine receptor 4 (CXCR4)-interacting agents that inhibit or stimulate, either competitively or allosterically, the intracellular Ca2+ release initiated by CXCR4 activation.

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include ...

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...