The authors thank all study participants and the UAB CCTS Bionutrition Core and UAB High Resolution Imaging Service Center for their contributions. This work was supported by NIH grants DK106284 and DK123542 (TM), and UL1TR003096 (National Center for Advancing Translational Sciences).

All experiments outlined in this work were approved by the University of Alabama at Birmingham (UAB) Institutional Review Board. Healthy adults (33.6 &#177; 3.3 years old; n=10) were enrolled in the study if they had a normal blood comprehensive metabolic panel, non-tobacco users, non-pregnant, a BMI between 20-30 kg/m2, and free of chronic medical conditions or acute illnesses. Healthy participants signed a written informed consent form prior to the start of the study.
1. Clinical protocol and urine collection
<ol>
	<li>Have participants consume a low oxalate diet prepared by the UAB Center for Clinical and Translational Sciences Bionutrition Core for 3 days and fast overnight before collecting their urine (24-hour sample).</li>
	<li>The following day, have participants return their 24-hour urine sample (pre-oxalate) before consuming an oxalate load (smoothie containing fruits and vegetables, ~8 mM oxalate). Have participants subsequently collect their urine for 24 hours (post-oxalate sample) and return their urine the following day.</li>
	<li>Maintain all urine samples at room temperature (RT) prior to processing as described below and shown in Figure 1.</li>
</ol>
2. Urine Processing
NOTE: All materials and equipment used are listed in Table of Materials. 
CAUTION: Wear personal protective equipment at all times while handling clinical samples and reagents. Specifically, gloves, face and eye shields, respiratory protection, and protective clothing.
<ol>
	<li>Measure and record urine pH and volume. Mix thoroughly prior to adding 50 mL of urine into a labeled sterile 50 mL conical tube.</li>
	<li>Centrifuge sample at 1200 x g for 10 min at RT using a benchtop centrifuge. 
	NOTE: Keep the sample at RT to prevent further crystal formation as cooler temperatures can promote crystallization.</li>
	<li>Discard the supernatant and wash and resuspend the pellet again with 5 mL of 100% ethanol. Centrifuge the sample at 1200 x g for 10 minutes at RT using a benchtop centrifuge.</li>
	<li>Discard the supernatant and resuspend the pellet in 1 mL of 100% ethanol. Store the sample at -20 &#176;C for later processing OR stain the sample as described below. 
	&#8203;NOTE: There is no significant difference in data points (i.e., particle size/concentration) between stored or freshly stained samples.</li>
</ol>
3. Nanoparticle Tracking Analysis (NTA)
<ol>
	<li>Sample Preparation
	<ol>
		<li>Gold nanoparticles: Use gold nanoparticles to optimize settings on the instrument. Dilute 100 nm sized gold nanoparticles 1:1000 in ultrapure water.</li>
		<li>Human Urine: Dilute urine samples 20 times in water prior to staining with 5 mM Fluo-4 AM (a calcium fluorescence dye) for 30 min in the dark. Analyze the samples using NTA.</li>
		<li>Prepare Calcium Oxalate (CaOx) crystals as previously described29. Dilute 10 mM stock solution (14.6 mg in 10 mL of water) to 50 &#181;M in water and stain the diluted samples using 5 mM Fluo-4 AM for 30 min in dark prior to analysis.</li>
		<li>Calcium Phosphate (CaP) crystals: Dilute 10 mM stock solution (50.4 mg in 10 mL of water) to 50 &#181;M in water and stain the diluted samples using 5 mM Fluo-4 AM for 30 min in dark prior to NTA analysis.</li>
	</ol>
	</li>
	<li>Instrument Set-up, Camera Settings, and Data Collection 
	NOTE: The computer and instrument setup used for this method are shown in Figure 2.
	<ol>
		<li>Turn on the computer and then the instrument. Open the software and turn on the camera.</li>
		<li>Once the software window is open, click the capture icon in the top left corner on the window to start the capture mode. The camera initialization takes a few seconds.</li>
		<li>Clean the platform by first pumping air into it using a 1 mL syringe until the platform appears clean. Gently add water to the apparatus 2-3 times using another 1 mL syringe to remove any air bubbles. 
		&#8203;NOTE: Look for any air bubbles in the platform as well as in the tubing. It is important to not have bubbles throughout the apparatus prior to and while running samples. If bubbles are present, clean the platform again with air and water.</li>
		<li>Once the platform is clean, add water to check for any contamination on the surface by viewing the camera. Next, add gold nanoparticles&#160;as a control to the sample loading pump injector to set up the instrument.</li>
		<li>Adjust the camera level on the screen or on the knob to the right side of the instrument until the image starts to display colored pixels and then reduce the camera level.</li>
		<li>Then adjust the screen to optimize the image. Left-click the mouse button on the video image. Hold the left mouse button and drag image up and down to get the entire view. 
		&#8203;NOTE: The normal camera lens and filter is used to assess gold nanoparticles and unstained samples.</li>
		<li>Set up the infusion speed and focus the camera so that the gold nanoparticles are visible on the camera screen. Set the infusion speed to high (i.e., 500 &#181;L/min) for initial set up to ensure the gold nanoparticles are detected. Once detected, reduce the speed to 50 &#181;L/min.</li>
		<li>Adjust the camera level to visualize the particles. For unstained samples, adjust the screen gain at level 5 to achieve the camera focus, and set the camera level at 8. Once the focus is set, record the sample (i.e., 1 measurement for 60 seconds only). 
		&#8203;NOTE: The focus and continuous flow speed are important for obtaining clear and sharp images of the particles for counting.</li>
		<li>After optimization, clean the apparatus again with water before assessing samples. View the camera to ensure that the tubing is clean and particles are not present. 
		&#8203;NOTE: Wash the chamber between each sample until no particles are detected by the camera.</li>
		<li>To analyze stained samples, adjust the camera to the filter position containing the suitable fluorescent filter. Load diluted and stained samples onto the sample loading pump injector and reduce the speed to 20 &#181;L/min for analysis of the sample.</li>
		<li>Next adjust the screen gain and camera level as these are important parameters. For stained (fluorescent) samples, set the screen gain to 5 and the camera level at level 13. 
		&#8203;NOTE: These parameters will vary based on the sample type and every sample will need to be optimized to gain focus.</li>
		<li>Use standard measurement to measure the samples for 5 captures per sample where one capture-duration is for 60 seconds.</li>
		<li>Save and store data after each measurement. The software will save image and video files for each measurement. The software provides output data (e.g., crystal size: 10 nM - 1000 nM and concentration) in both excel and pdf formats.</li>
		<li>Calculate the average number of nanoparticles for all 5 readings for each individual sample. Analyze the data using standard deviation or standard error of the mean and use t-tests for paired analysis.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Fogazzi, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystalluria:+a+neglected+aspect+of+urinary+sediment+analysis.">Crystalluria: a neglected aspect of urinary sediment analysis.</a> Nephrology, Dialysis, Transplantation. 11 (2), 379-387 (1996).</li><li>Kuo, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urine+calcium+and+volume+predict+coverage+of+renal+papilla+by+Randall's+plaque.">Urine calcium and volume predict coverage of renal papilla by Randall's plaque.</a> Kidney International. 64 (6), 2150-2154 (2003).</li><li>Robertson, W. G., Peacock, M., Nordin, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+crystalluria+in+recurrent+renal-stone+formers.">Calcium crystalluria in recurrent renal-stone formers.</a> Lancet. 2 (7610), 21-24 (1969).</li><li>Robertson, W. G., Peacock, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+oxalate+crystalluria+and+inhibitors+of+crystallization+in+recurrent+renal+stone-formers.">Calcium oxalate crystalluria and inhibitors of crystallization in recurrent renal stone-formers.</a> Clinical Science. 43 (4), 499-506 (1972).</li><li>Hallson, P. C., Rose, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+urinary+test+for+stone+"activity".">A new urinary test for stone "activity".</a> British Journal of Urology. 50 (7), 442-448 (1978).</li><li>Daudon, M., Hennequin, C., Boujelben, G., Lacour, B., Jungers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+crystalluria+determination+and+the+risk+of+recurrence+in+calcium+stone+formers.">Serial crystalluria determination and the risk of recurrence in calcium stone formers.</a> Kidney International. 67 (5), 1934-1943 (2005).</li><li>Baumann, J. M., Affolter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+crystalluria+to+kidney+stones,+some+physicochemical+aspects+of+calcium+nephrolithiasis.">From crystalluria to kidney stones, some physicochemical aspects of calcium nephrolithiasis.</a> World Journal of Nephrology. 3 (4), 256-267 (2014).</li><li>Patel, 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=Oxalate+induces+mitochondrial+dysfunction+and+disrupts+redox+homeostasis+in+a+human+monocyte+derived+cell+line.">Oxalate induces mitochondrial dysfunction and disrupts redox homeostasis in a human monocyte derived cell line.</a> Redox Biology. 15, 207-215 (2018).</li><li>Khan, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+renal+epithelial+cells+in+the+initiation+of+calcium+oxalate+stones.">Role of renal epithelial cells in the initiation of calcium oxalate stones.</a> Nephron Experimental Nephrology. 98 (2), 55-60 (2004).</li><li>Mulay, S. R., 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=Calcium+oxalate+crystals+induce+renal+inflammation+by+NLRP3-mediated+IL-1beta+secretion.">Calcium oxalate crystals induce renal inflammation by NLRP3-mediated IL-1beta secretion.</a> Journal of Clinical Investigation. 123 (1), 236-246 (2013).</li><li>Umekawa, T., Chegini, N., Khan, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxalate+ions+and+calcium+oxalate+crystals+stimulate+MCP-1+expression+by+renal+epithelial+cells.">Oxalate ions and calcium oxalate crystals stimulate MCP-1 expression by renal epithelial cells.</a> Kidney International. 61 (1), 105-112 (2002).</li><li>Huang, M. Y., Chaturvedi, L. S., Koul, S., Koul, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxalate+stimulates+IL-6+production+in+HK-2+cells,+a+line+of+human+renal+proximal+tubular+epithelial+cells.">Oxalate stimulates IL-6 production in HK-2 cells, a line of human renal proximal tubular epithelial cells.</a> Kidney International. 68 (2), 497-503 (2005).</li><li>Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+tubular+epithelial+cell+injury,+apoptosis+and+inflammation+are+involved+in+melamine-related+kidney+stone+formation.">Renal tubular epithelial cell injury, apoptosis and inflammation are involved in melamine-related kidney stone formation.</a> Urological Research. 40 (6), 717-723 (2012).</li><li>Williams, J., Holmes, R. P., Assimos, D. G., Mitchell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+Mitochondrial+Function+in+Calcium+Oxalate+Stone+Formers.">Monocyte Mitochondrial Function in Calcium Oxalate Stone Formers.</a> Urology. 93, 221-226 (2016).</li><li>Balcke, 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=Transient+hyperoxaluria+after+ingestion+of+chocolate+as+a+high+risk+factor+for+calcium+oxalate+calculi.">Transient hyperoxaluria after ingestion of chocolate as a high risk factor for calcium oxalate calculi.</a> Nephron. 51 (1), 32-34 (1989).</li><li>Khan, S. R., Kok, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulators+of+urinary+stone+formation.">Modulators of urinary stone formation.</a> Frontiers in Bioscience. 9, 1450-1482 (2004).</li><li>Rodgers, A., Allie-Hamdulay, S., Jackson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+action+of+citrate+in+urolithiasis+explained+by+chemical+speciation:+increase+in+pH+is+the+determinant+factor.">Therapeutic action of citrate in urolithiasis explained by chemical speciation: increase in pH is the determinant factor.</a> Nephrology, Dialysis, Transplantation. 21 (2), 361-369 (2006).</li><li>Verplaetse, H., Verbeeck, R. M., Minnaert, H., Oosterlinck, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solubility+of+inorganic+kidney+stone+components+in+the+presence+of+acid-base+sensitive+complexing+agents.">Solubility of inorganic kidney stone components in the presence of acid-base sensitive complexing agents.</a> European Urology. 11 (1), 44-51 (1985).</li><li>Frochot, V., Daudon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+value+of+crystalluria+and+quantitative+morphoconstitutional+analysis+of+urinary+calculi.">Clinical value of crystalluria and quantitative morphoconstitutional analysis of urinary calculi.</a> International Journal of Surgery. 36, 624-632 (2016).</li><li>Grover, P. K., Thurgood, L. A., Wang, T., Ryall, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+intracrystalline+and+surface-bound+proteins+on+the+attachment+of+calcium+oxalate+monohydrate+crystals+to+renal+cells+in+undiluted+human+urine.">The effects of intracrystalline and surface-bound proteins on the attachment of calcium oxalate monohydrate crystals to renal cells in undiluted human urine.</a> BJU International. 105, 708-715 (2010).</li><li>Bader, C. A., Chevalier, A., Hennequin, C., Jungers, P., Daudon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+aspects+of+spontaneous+crystalluria+studies+in+calcium+stone+formers.">Methodological aspects of spontaneous crystalluria studies in calcium stone formers.</a> Scanning Microscopy. 8 (2), 215-231 (1994).</li><li>Daudon, M., Cohen-Solal, F., Jungers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Eurolithiasis. 9th European Symposium on Urolithiasis. , 261-263 (2001).</li><li>Werness, P. G., Bergert, J. H., Smith, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystalluria.">Crystalluria.</a> Journal of Crystal Growth. 53 (1), 166-181 (1981).</li><li>Fan, J., Chandhoke, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+crystalluria+in+freshly+voided+urines+of+recurrent+calcium+stone+formers+and+normal+individuals+using+a+new+filter+technique.">Examination of crystalluria in freshly voided urines of recurrent calcium stone formers and normal individuals using a new filter technique.</a> Journal of Urology. 161 (5), 1685-1688 (1999).</li><li>Sun, X. Y., Ouyang, J. M., Yu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape-dependent+cellular+toxicity+on+renal+epithelial+cells+and+stone+risk+of+calcium+oxalate+dihydrate+crystals.">Shape-dependent cellular toxicity on renal epithelial cells and stone risk of calcium oxalate dihydrate crystals.</a> Scientific Reports. 7 (1), 7250 (2017).</li><li>He, J. Y., Deng, S. P., Ouyang, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology,+particle+size+distribution,+aggregation,+and+crystal+phase+of+nanocrystallites+in+the+urine+of+healthy+persons+and+lithogenic+patients.">Morphology, particle size distribution, aggregation, and crystal phase of nanocrystallites in the urine of healthy persons and lithogenic patients.</a> IEEE Trans Nanobioscience. 9 (2), 156-163 (2010).</li><li>Gao, 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=Comparison+of+Physicochemical+Properties+of+Nano-+and+Microsized+Crystals+in+the+Urine+of+Calcium+Oxalate+Stone+Patients+and+Control+Subjects.">Comparison of Physicochemical Properties of Nano- and Microsized Crystals in the Urine of Calcium Oxalate Stone Patients and Control Subjects.</a> Journal of Nanomaterials. 2014, 9 (2014).</li><li>Gavin, 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=Novel+Methods+of+Determining+Urinary+Calculi+Composition:+Petrographic+Thin+Sectioning+of+Calculi+and+Nanoscale+Flow+Cytometry+Urinalysis.">Novel Methods of Determining Urinary Calculi Composition: Petrographic Thin Sectioning of Calculi and Nanoscale Flow Cytometry Urinalysis.</a> Scientific Reports. 6, 19328 (2016).</li><li>Kumar, 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=Dietary+Oxalate+Induces+Urinary+Nanocrystals+in+Humans.">Dietary Oxalate Induces Urinary Nanocrystals in Humans.</a> Kidney International Reports. 5 (7), 1040-1051 (2020).</li><li>Carr, B., Hole, P., Malloy, A., Nelson, P., Smith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+nanoparticle+tracking+analysis+in+nanoparticle+research--A+mini-review.">Applications of nanoparticle tracking analysis in nanoparticle research--A mini-review.</a> European Journal of Parenteral Sciences and Pharmaceutical Sciences. 14 (2), 45 (2009).</li><li>Dragovic, R. A., 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=Sizing+and+phenotyping+of+cellular+vesicles+using+Nanoparticle+Tracking+Analysis.">Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis.</a> Nanomedicine: Nanotechnology, Biology, and Medicine. 7 (6), 780-788 (2011).</li><li>Dragovic, R. A., 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=Isolation+of+syncytiotrophoblast+microvesicles+and+exosomes+and+their+characterisation+by+multicolour+flow+cytometry+and+fluorescence+Nanoparticle+Tracking+Analysis.">Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence Nanoparticle Tracking Analysis.</a> Methods. 87, 64-74 (2015).</li><li>Gercel-Taylor, C., Atay, S., Tullis, R. H., Kesimer, M., Taylor, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+analysis+of+circulating+cell-derived+vesicles+in+ovarian+cancer+patients.">Nanoparticle analysis of circulating cell-derived vesicles in ovarian cancer patients.</a> Analytical Biochemistry. 428 (1), 44-53 (2012).</li><li>Minta, A., Kao, J. P., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+indicators+for+cytosolic+calcium+based+on+rhodamine+and+fluorescein+chromophores.">Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores.</a> Journal of Biological Chemistry. 264 (14), 8171-8178 (1989).</li><li>Harkins, A. B., Kurebayashi, N., Baylor, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+myoplasmic+free+calcium+in+frog+skeletal+muscle+fibers+estimated+with+fluo-3.">Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo-3.</a> Biophysical Journal. 65 (2), 865-881 (1993).</li><li>Hernandez-Santana, A., Yavorskyy, A., Loughran, S. T., McCarthy, G. M., McMahon, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+in+the+detection+of+calcium-containing+microcrystals+in+synovial+fluid.">New approaches in the detection of calcium-containing microcrystals in synovial fluid.</a> Bioanalysis. 3 (10), 1085-1091 (2011).</li><li>Tong, M., Brown, O. S., Stone, P. R., Cree, L. M., Chamley, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+speed+alters+the+apparent+size+and+concentration+of+particles+measured+using+NanoSight+nanoparticle+tracking+analysis.">Flow speed alters the apparent size and concentration of particles measured using NanoSight nanoparticle tracking analysis.</a> Placenta. 38, 29-32 (2016).</li><li>Maas, S. 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=Possibilities+and+limitations+of+current+technologies+for+quantification+of+biological+extracellular+vesicles+and+synthetic+mimics.">Possibilities and limitations of current technologies for quantification of biological extracellular vesicles and synthetic mimics.</a> Journal of Controlled Release. 200, 87-96 (2015).</li><li>Hole, 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=Interlaboratory+comparison+of+size+measurements+on+nanoparticles+using+nanoparticle+tracking+analysis+(NTA).">Interlaboratory comparison of size measurements on nanoparticles using nanoparticle tracking analysis (NTA).</a> Journal of Nanoparticle Research. 15, 2101 (2013).</li><li>Tomlinson, P. R., 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+of+distinct+circulating+exosomes+in+Parkinson's+disease.">Identification of distinct circulating exosomes in Parkinson's disease.</a> Annals of Clinical and Translational Neurology. 2 (4), 353-361 (2015).</li></ol>

The authors declare no conflicts of interest.

NTA has been used in the present study to assess nanocrystals in human urine using a calcium binding probe, Fluo-4 AM. There is no standard method available to detect nanocrystals in the urine. Some research groups have detected nanocrystals in the urine and relied on the use of extensive protocols or methods that are limited in their ability to quantify the samples27,28. This study shows a specific and sensitive method for detecting calcium containing nanocrysta...

Urinary crystals form when urine becomes supersaturated with minerals. This can occur in healthy individuals but is more common in individuals with kidney stones1. The presence and accumulation of urinary crystals can increase one&#39;s risk of developing a kidney stone. Specifically, this occurs when crystals bind to Randall&#39;s plaque, nucleate, accumulate, and grow over time2,3,4. Crystalluria precedes kidney stone formation and assessment of crystalluria may have predictive value in kidney stone formers3,5. Specifically, crystalluria has been suggested to be useful to predict the risk of stone recurrence in patients with a history of calcium oxalate containing stones6,7.
Crystals have been reported to negatively impact renal epithelial and circulating immune cell function8,9,10,11,12,13. It has been previously reported that circulating monocytes from calcium oxalate (CaOx) kidney stone formers have suppressed cellular bioenergetics compared to healthy individuals14. In addition, CaOx crystals reduce cellular bioenergetics and disrupt redox homeostasis in monocytes8. Consumption of meals rich in oxalate may cause crystalluria which could lead to renal tubule damage and alter the production and function of urinary macromolecules that are protective against kidney stone formation15,16. Several studies have demonstrated that urinary crystals can vary in shape and size depending on the pH and temperature of the urine17,18,19. Further, urinary proteins have been shown to modulate crystal behavior20. Daudon et al.19, proposed that crystalluria analysis could be helpful in the management of patients with kidney stone disease and in assessing their response to therapies. A few conventional methods currently available to evaluate the presence of crystals include polarized microscopy21,22, electron microscopy23, particle counters3, urine filtration24, evaporation3,5 or centrifugation21. These studies have provided valuable insight to the kidney stone field regarding crystalluria. However, a limitation of these methods has been the inability to visualize and quantify crystals less than 1 &#181;m in size. Crystals of this size may influence the growth of CaOx stones by attaching to Randall&#39;s plaque.
Nanocrystals have been shown to cause extensive injury to renal cells compared to larger microcrystals25. The presence of nanocrystals has been reported in urine using a nanoparticle analyzer26,27. Recent studies have used fluorescently labeled bisphosphate probes (alendronate-fluorescein/alendronate-Cy5) to examine nanocrystals using nanoscale flow cytometry28. The limitation of this dye is that it is not specific and will bind to almost all types of stones except cysteine. Thus, accurately assessing the presence of nanocrystals in individuals may be an effective tool to diagnose crystalluria and/or predict stone risk. The purpose of this study was to detect and quantify calcium containing nanocrystals (&#60;1 &#181;m in size) using nanoparticle tracking analysis (NTA). To achieve this, NTA technology was used in combination with a calcium binding fluorophore, Fluo-4 AM to detect and quantify calcium containing nanocrystals in the urine of healthy adults.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Benchtop Centrifuge</td>
			<td>Jouan Centrifuge</td>
			<td>CR3-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Oxalate monohydrate</td>
			<td>Synthesized in the lab as previously described29.</td>
			<td> </td>
			<td>Store at RT; Stock 10 mM</td>
		</tr>
		<tr>
			<td>Calcium Phosphate crystals (hydroxyapatite nanopowder)</td>
			<td>Sigma</td>
			<td>677418</td>
			<td>Store at RT; Stock 10 mM</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fischer Scientific</td>
			<td>AC615095000</td>
			<td>Store at RT; Stock 100%</td>
		</tr>
		<tr>
			<td>Fluo-4 AM*</td>
			<td>AAT Bioquest, Inc.</td>
			<td>20550</td>
			<td>Store at Freezer (-20&#176;C); Stock 5 mM</td>
		</tr>
		<tr>
			<td>Gold Nanoparticles</td>
			<td>Sigma</td>
			<td>742031</td>
			<td>Store at 2-8&#176;C</td>
		</tr>
		<tr>
			<td>NanoSight Instrument</td>
			<td>Malvern Instruments, UK</td>
			<td>NS300</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>98-4730</td>
			<td></td>
		</tr>
		<tr>
			<td>Virkon Disinfectant</td>
			<td>LanXESS Energizing Company, Germany</td>
			<td>LSP</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">*Fluorescence dyes are light sensitive; stock and aliquots should be stored in the dark at -20&#176;C.</td>
		</tr>
	</tbody>
</table>

estimation of urinary nanocrystals in humans using calcium fluorophore labeling and nanoparticle tracking analysis

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) crystals. Crystalluria occurs when urine becomes supersaturated with minerals (e.g., calcium, oxalate, phosphate) and precedes kidney stone formation. Standard methods to assess crystalluria in stone formers include microscopy, filtration, and centrifugation. However, these methods primarily detect microcrystals and not nanocrystals. Nanocrystals have been suggested to be more harmful to kidney epithelial cells than microcrystals in vitro. Here, we describe the ability of Nanoparticle Tracking analysis (NTA) to detect human urinary nanocrystals. Healthy adults were fed a controlled oxalate diet prior to drinking an oxalate load to stimulate urinary nanocrystals. Urine was collected for 24 hours before and after the oxalate load. Samples were processed and washed with ethanol to purify samples. Urinary nanocrystals were stained with the calcium binding fluorophore, Fluo-4 AM. After staining, the size and count of nanocrystals were determined using NTA. The findings from this study show NTA can efficiently detect nanocrystalluria in healthy adults. These findings suggest NTA could be a valuable early detection method of nanocrystalluria in patients with kidney stone disease.

The findings from this study show NTA can efficiently detect the mean size and concentration of calcium containing urinary nanocrystals in human urine. This was achieved by using the fluorophore, Fluo-4 AM, and nanoparticle tracking analysis. Fluo-4 AM was able to bind to both CaOx and CaP crystals. As shown in Figure 3A, CaOx crystals were determined to be between 50-270 nm in size and have a mean concentration of 1.26 x 109 particles/mL. CaP crystals were between 30-225 nm in si...

Watch this Scientific Journal Video about Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis at JoVE.com

Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis

The objective of this study was to determine whether nanoparticle tracking analysis (NTA) could detect and quantify urinary calcium containing nanocrystals from healthy adults. The findings from the current study suggest NTA could be a potential tool to estimate urinary nanocrystals during kidney stone disease.

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) ...

estimation-urinary-nanocrystals-humans-using-calcium-fluorophore

Research

JoVE Journal

Medicine

3.5K Views.  University of Alabama at Birmingham. The objective of this study was to determine whether nanoparticle tracking analysis (NTA) could detect and quantify urinary calcium containing nanocrystals from healthy adults. The findings from the current study suggest NTA could be a potential tool to estimate urinary nanocrystals during kidney stone disease. 

Video: Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes.
A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages 1. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo.
Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles 2-4. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images 5. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM-1 sec-1 strength4. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure 6. Our group has applied this agent in an &ldquo;off label&rdquo; use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.

We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena described in those patients for whom a brain autopsy has been requested. Although the advancements of biomedical sciences and neuroimaging have revolutionized the neuropsychiatric field, they have also generated the misleading idea that brain autopsies have only a confirmatory value. This false idea created a drastic reduction of autopsy rates and, consequently, a reduced possibility to perform more detailed and extensive neuropathological investigations, which are necessary to comprehend numerous normal and pathological aspects yet unknown of the human brain. The traditional inferential method of correlation between observed neuropsychiatric phenomena and corresponding localization/characterization of their possible neurohistological correlates continues to have an undeniable value. In the context of neuropsychiatric diseases, the traditional clinicopathological method is still the best possible methodology (and often the only available) to link unique neuropsychiatric features to their corresponding neuropathological substrates, since it relies specifically upon the direct physical assessment of brain tissues. The assessment of postmortem brains is based on brain cutting procedures that vary across different neuropathology centers. Brain cuttings are performed in a relatively extensive and systematic way based on the various clinical and academic contingencies present in each institution. A more anatomically inclusive and symmetric bi-hemispheric brain cutting methodology should at least be used for research purposes in human neuropathology to coherently investigate, in depth, normal and pathological conditions with the peculiarities of the human brain (i.e., hemispheric specialization and lateralization for specific functions). Such a method would provide a more comprehensive collection of neuropathologically well-characterized brains available for current and future biotechnological and neuroimaging techniques. We describe a symmetric bi-hemispheric brain cutting procedure for the investigation of hemispheric differences in human brain pathologies and for use with current as well as future biomolecular/neuroimaging techniques.

Organized brain cutting procedures are necessary to correlate specific neuropsychiatric phenomena with definitive neuropathologic diagnoses. Brain cuttings are performed differently based on various clinico-academic contingencies. This protocol describes a symmetric bihemispheric brain cutting procedure to investigate hemispheric differences in human brain pathologies and to maximize current and future biomolecular/neuroimaging techniques.

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena ...

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120&#176;/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.

This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...

A Model to Simulate Clinically Relevant Hypoxia in Humans

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2).

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...

Comparing Bibliometric Analysis Using PubMed, Scopus, and Web of Science Databases

Literature databases (i.e., PubMed, Scopus, and Web of Science) differ in terms of their coverage, focus, and the tool they provide. PubMed focuses mainly on life sciences and biomedical disciplines, whereas Scopus and Web of Science are multidisciplinary. The protocol described in the current study was used to search for publications from Jordanian authors in the years 2013-2017. In this protocol, how to use each database to conduct this type of search is explained in detail. A Scopus search resulted in the highest number of documents (11,444 documents), followed by a Web of Science search (10,943 documents). PubMed resulted in a smaller number of documents due to its narrower scope and coverage (4,363 documents). The results also show a yearly trend in: (1) the number of publications, (2) the disciplines that have the most publications, (3) the countries of collaboration, and (4) the number of open access publications. In contrast, PubMed has a sophisticated keyword optimization service (i.e., Medical Subject Heading, or MeSH), while both Scopus and Web of Science provide search analysis tools that can produce representative figures. Finally, the features of each database are explained in detail and several indices that can be extracted using the search results are provided. This study provides a base for using literature databases for bibliometric analysis.

Literature databases are commonly used to assess publications in a certain subject, discipline, country, or region of the world, a practice known as bibliometric analysis. The current protocol details how to use PubMed, Scopus, and Web of Science databases to do bibliometric analysis.

Literature databases (i.e., PubMed, Scopus, and Web of Science) differ in terms of their coverage, focus, and the tool they provide. PubMed focuses mainly ...

Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and no new nephrons are formed post-birth. Nephron number refers to the total number of nephrons measured at any point in time post-birth. Both genetic and environmental factors influence both nephron endowment and number. Understanding how specific genes or factors influence the process of nephrogenesis and nephron loss or demise is important as individuals with lower nephron endowment or number are thought to be at a higher risk of developing renal or cardiovascular disease. Understanding how environmental exposures over the course of a person's lifetime affects nephron number will also be vital in determining future disease risk. Thus, the ability to assess whole kidney nephron number quickly and reliably is a basic experimental requirement to better understand mechanisms that contribute to or promote nephrogenesis or nephron loss. Here, we describe the acid maceration method for the estimation of whole kidney nephron number based on the procedure described by Damadian, Shawayri, and Bricker, with slight modifications. The acid maceration method provides fast and reliable estimates of nephron number (as assessed by counting glomeruli) that are within 5% of those determined using more advanced, albeit expensive, methods such as magnetic resonance imaging. Moreover, the acid maceration method is an excellent high-throughput method to assess nephron number in large numbers of samples or experimental conditions.

Estimates of whole kidney nephron number are important clinically and experimentally, as there is an inverse association between nephron number and an enhanced risk of renal and cardiovascular disease.Herein, the use of the acid maceration method, which provides fast and reliable estimates of whole kidney nephron number, is demonstrated.

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...